Marek&#39;s disease virus genes and their use in vaccines for protection against marek&#39;s disease

ABSTRACT

A nucleotide sequence encoding the gp82 polypeptide of Marek&#39;s disease virus is disclosed. Also disclosed are recombinant viruses which are useful as vaccines for protecting against Marek&#39;s Disease, preferably containing two more genes encoding Marek&#39;s Disease Virus antigens such as glyprotein B and glycoprotein gp82, under the control of a poxvirus promoter within a region of the DNA of fowlpox virus which is not essential for virus growth. Also provided is a vaccine exhibiting a synergistic immunoprotective effect, comprising a recombinant fowlpox virus expressing Marek&#39;s Disease Virus gB protein in combination with turkey herpesvirus. A method of immunizing poultry, comprising administering any of the disclosed vaccines, is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of copending application Ser.No. 08/709,731, filed on Sep. 9, 1996 which is a continuation of PCTApplication No. PCT/US96/11360 filed on Jul. 5, 1996 which designatedthe United States, and which is a continuation-in-part of U.S. Ser. No.08/499,474 filed on Jul. 7, 1995, priority of which applications isclaimed under 35 U.S.C. §120. The entire contents of all of theseapplications are incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a gene derived from Marek'sdisease virus having a unique nucleotide sequence, recombinant virusescontinuing this gene, poultry vaccines utilizing this gene, andrecombinant fowlpox vaccines that exhibit a syngeristic effect inprotecting against Marek's disease.

[0004] 2. Description of Related Art

[0005] Marek's disease (MD) is a highly contagious neoplastic disease ofdomestic chickens that affects chickens worldwide and causes highmortality and condemnation if chickens are not vaccinated at one day ofage. MD is caused by a highly cell-associated oncogenic herpesvirusknown as Marek's disease virus (MDV).

[0006] A number of live virus cell-associated vaccines are availablethat protect chickens against MD. These vaccines are maintained andadministered in delicate, cell-associated form. The vaccines needspecial handling, and must be stored and transported in a frozen statein liquid nitrogen in order to maintain their viability and efficacy.These existing vaccines must be maintained and administered incell-associated form, a condition that is costly and cumbersome.

[0007] The known vaccines contain the entire MDV genome, includingsequences related to induction of pathogenesis. Although the existingvaccines against MD are either attenuated or are naturally apathogenic,viral mutation is known to occur in herpesviruses, and there is apossibility that virulent pathogenic mutants may emerge in suchvaccines. Such mutants could be less effective and even harmful.

[0008] Churchill et al. (Nature 221:744-747 (1969)) and Okazaki et al.(Avian Dis. 14:413-429 (1970)) developed the first effective and safevaccines against MD. These vaccines have been in use for the last 20years, and have reduced losses to the poultry industry worldwide. Othercandidate vaccines based on serotype 2 naturally apathogenic MDV (Schatet al. J. Natl. Cancer Inst. 60: 1075-1082 (1978)), or newly attenuatedserotype 1 MDV (Rispens et al. Avian Dis. 16:108-125 (1972)), andcombinations of these viruses as bivalent vaccines (Witter Avian Dis.31:252-257 (1987)), have helped provide better protection against MD.All these vaccines, except the herpesvirus of turkeys (HVT) vaccine,require storage and transportation in a frozen state in liquid nitrogenand have to be administered as infected cells, which calls for carefulprocedures to prevent inactivation of the vaccine. Even in the case ofHVT vaccine, cell-associated viruses have been most widely used becausethey are more effective than cell-free virus in the presence of maternalantibodies (Witter et al. Avian Pathol. 8:145-156 (1978)).

[0009] Recombinant DNA technology has facilitated the construction ofrecombinant vaccines that contain only those desired viral genes or geneproducts that induce immunity without exposing the animal to genes thatmay induce pathological disorders. Pox viruses, including avipox virus,especially fowlpox virus (FPV), provide excellent models for suchvaccines. These viruses have a large DNA molecule with numerousnon-essential regions that permit the insertion of several immunogenicgenes into the same virus for the purpose of creating multivalentvaccines. These multivalent vaccines may induce cell-medicateod as wellas antibody-mediated immune response in a vaccinated host. Vacciniavirus (VV) has been used extensively for this purpose, and a number ofVV recombinants have been constructed that express a variety of foreigngenes, including those that elicit neutralizing antibodies againstglycoproteins of herpes simplex virus (HSV) type 1 (Blacklaws et al.Virology 177:727-736 (1990)). Similarly, there are number of reportsdescribing the expression of foreign genes by recombinant FPV (Boyle etal. Virus Res. 10:343-356 (1988) and Ogawa et al. Vaccine 8:486-490(1990)). Recently, we demonstrated that the recFPVgB protected chickensagainst MDV challenge (Nazerian et al. J. Virol. 66:1409-1413 (1992)).

[0010] MDV homologues of the HSV genes coding for glycoproteins B, C, D,H, and I, E, L (gB, gC, gD, gH and gI, gE, gL) have recently been clonedand sequenced (Coussens et al. J. Virol. 62:2373-2379 (1988); Ross etal. J. Gen. Virol. 70:1789-1804 (1989); Ross et al. J. Gen. Virol.72:939-947 (1991); Ross et al., International Publication No. WO90/02803 (1990); Brunovskis and Velicer, Virology 206:324-338 (1995);and Yoshida et al. Virology 204:414-419 (1994)).

SUMMARY OF THE INVENTION

[0011] The present inventors have shown that gB is an important gene forprotective immunity against MD (Nazerian et al. J. Virol. 3066:1409-1413 (1992)). Whittaker et al. (1992) reported that Equineherpesvirus type 1 (EHV-1) gene 28 encodes a glycoprotein, gp300, thatis homologous to the HSV-1 UL32, and functions in EHV-1 in cell-to-cellfusion processes. We postulated that if such a homologous gene existedin MDV, it may function in cell to cell fusion since MDV is acell-associated virus. Recently, we identified and sequenced an MDV genehomologous to HSV-1 UL32, and identified an O-linked glycoprotein, gp82,in MDV-infected cells belonging to a class of membrane proteins (Lee,unpublished data).

[0012] The present invention relates to the MDV UL32 gene encoding amembrane glycoprotein. The DNA sequence of the UL32 gene is shown in SEQID NO:1 in the attached Sequence Listing. The present inventiontherefore relates to this sequence, which encodes a protein inaccordance with the degeneracy of the genetic code, preferably in acloned, isolated, or purified form, and biologically functional variantsthereof. The present invention also relates to recombinant DNA moleculescomprising the UL32 sequence.

[0013] The present invention also relates to novel recombinant viralvaccines, such as recombinant FPV, HVT (herpesvirus of turkeys), MDV,and ILTV (infectious laryngotracheitis virus), that contain the novelUL32 gene encoding membrane glycoprotein gp82 of MDV. Preferably, thevaccine is based on a recombinant FPV containing the UL32 gene of MDV.More preferably, the recombinant FPV contains an additional gene, i.e.,the gB gene, encoding gp100, gp60 and gp49, which provides a synergisticeffect in protecting against MD in chickens. As shown below, recombinantFPV expressing UL32 is effective against MD. The sequence and theeffectiveness of gB as a vaccine has been described in U.S. Pat. No.5,369,025. The expression of these two genes in cells results in anunexpectedly strong synergistic protective effect against MD in thenatural host (chickens). In addition, the vaccine also protects againstfowlpox.

[0014] The present invention also relates to recombinant FPV vaccinesagainst MD in which the gB gene of MDV or the UL32 gene of MDV or othergenes such as those coding for glycoprotein E homologue, glycoprotein Ihomologue, and other glycoproteins from different serotypes of MDV areinserted into FPV for the purpose of creating a broad-spectrum vaccineeffective against several isolates of MDV.

[0015] The present invention also relates to a cell-free vaccine againstMD containing recombinant (rec) FPV that can be lyophilized, stored, andused under normal conditions, thereby obviating costly and laboriousprocedures of storing the vaccine in liquid nitrogen, delicate handling,and administering which are necessary with existing cell-associated MDvaccines. For example, after lyophilization, the vaccine of the presentinvention can be stored, handled, and transported at ambient temperature(20-22° C.), and stored at 4° C. for prolonged periods of time. Thevaccine can also be stored in a frozen state wherein the cell-freerecombinant virus is present in an aqueous solution which is frozen andstored at, for example, −20° C. or −70° C.

[0016] Accordingly, it is an object of the present invention to providean isolated, purified DNA molecule comprising the nucleotide sequenceshown in SEQ ID NO:1, or a nucleotide sequence biologically functionallyequivalent thereto.

[0017] Another object of the present invention is to provide anisolated, purified DNA molecule comprising a nucleotide sequenceencoding a polypeptide having the amino acid sequence shown in SEQ IDNO:2, or encoding a polypeptide biologically functionally equivalentthereto.

[0018] Another object of the present invention is to provide anisolated, purified polypeptide having the amino acid sequence shown inSEQ ID NO:2, or a polypeptide biologically functionally equivalentthereto.

[0019] Another object of the present invention is to provide arecombinant vector comprising the aforementioned DNA molecules.

[0020] Another object of the present invention is to provide arecombinant virus that expresses said DNA molecules. The recombinantvirus can further express a nucleotide sequence encoding at least oneantigen of an avian pathogen, or a nucleotide sequence biologicallyfunctionally equivalent thereto.

[0021] Another object of the present invention is to provide arecombinant virus that expresses a DNA sequence encoding a membraneglycoprotein of Marek's Disease virus.

[0022] Another object of the present invention is to provide a vaccinecomposition, comprising a member selected from the group consisting of:

[0023] an isolated, purified DNA molecule comprising the nucleotidesequence shown in SEQ ID NO:1, or a nucleotide sequence biologicallyfunctionally equivalent thereto;

[0024] an isolated, purified DNA molecule comprising a nucleotidesequence encoding a polypeptide having the amino acid sequence shown inSEQ ID NO:2, or a polypeptide biologically functionally equivalentthereto;

[0025] an isolated, purified polypeptide having the amino acid sequenceshown in SEQ ID NO:2. or a polypeptide biologically functionallyequivalent thereto;

[0026] a recombinant vector comprising an isolated, purified DNAmolecule comprising the nucleotide sequence shown in SEQ ID NO:1, or anucleotide sequence biologically functionally equivalent thereto;

[0027] a recombinant vector comprising an isolated, purified DNAmolecule comprising a nucleotide sequence encoding a polypeptide havingthe amino acid sequence as shown in SEQ ID NO:2, or a polypeptidebiologically functionally equivalent thereto;

[0028] a recombinant virus that expresses an isolated, purified DNAmolecule comprising the nucleotide sequence shown in SEQ ID NO:1, or anucleotide sequence biologically functionally equivalent thereto;

[0029] a recombinant virus that expresses an isolated, purified DNAmolecule comprising a nucleotide sequence encoding a polypeptide havingthe amino acid sequence as shown in SEQ ID NO:2, or a polypeptidebiologically functionally equivalent thereto; and

[0030] a recombinant virus that expresses an isolated, purified DNAmolecule comprising the nucleotide sequence shown in SEQ ID NO:1, or anucleotide sequence biologically functionally equivalent thereto, andwhich further expresses a nucleotide sequence encoding at least oneantigen of an avian pathogen, and a pharmaceutically acceptable carrier.

[0031] A vaccine composition of the present invention is most preferablyeffective for immunizing the vaccinated subject even in the presence oftransfer antibodies conferred from the mother of the subject.

[0032] Yet another object of the present invention is to provide avaccine composition, comprising a member selected from the groupconsisting of:

[0033] a recombinant virus that expresses an isolated, purified DNAmolecule comprising the nucleotide sequence shown in SEQ ID NO:1, or anucleotide sequence biologically functionally equivalent thereto, and gBantigen of Marek's disease virus or a polypeptide biologicallyfunctionally equivalent thereto, and

[0034] a virus that expresses an isolated, purified DNA moleculecomprising a nucleotide sequence encoding a polypeptide having the aminoacid sequence as shown in SEQ ID NO:2, or a polypeptide biologicallyfunctionally equivalent thereto, and gB antigen of Marek's disease virusor a polypeptide biologically functionally equivalent thereto, and apharmaceutically acceptable carrier.

[0035] Another object of the present invention is to provide a vaccinecomposition, comprising isolated, purified nucleotide sequences encodingantigens from avian pathogens, wherein said vaccine composition exhibitsan immunoprotective effect greater than the sum of the individualimmunoprotective effects of vaccine compositions individually comprisingeach of said isolated, purified nucleotide sequences encoding antigensfrom avian pathogens, and a pharmaceutically acceptable carrier.

[0036] Another object of the present invention is to provide a vaccinecomposition, comprising a recombinant virus expressing an isolated,purified nucleotide sequence encoding a Marek's disease viruspolypeptide or a biologically functionally equivalent polypeptide, incombination with a herpesvirus, wherein said vaccine compositionexhibits an immunoprotective effect greater than the sum of theindividual immunoprotective effects of vaccine compositions individuallycomprising each of said viruses, and a pharmaceutically acceptablecarrier.

[0037] A still further object of the present invention is to provide avaccine composition, comprising a member selected from the groupconsisting of:

[0038] a DNA molecule having the sequence shown in SEQ ID No:1 or asequence biologically functionally equivalent thereto;

[0039] a recombinant vector that contains a DNA molecule having thesequence shown in SEQ ID NO:1 or a sequence biologically functionallyequivalent thereto;

[0040] a recombinant virus or viruses that contains a DNA moleculehaving the sequence shown in SEQ ID NO:1 or a sequence biologicallyfunctionally equivalent thereto, as well as a DNA sequence encoding atleast one antigen of an avian pathogen, or a nucleotide sequencebiologically functionally equivalent thereto; and

[0041] a polypeptide having the amino acid sequence shown in SEQ IDNO:2, or a polypeptide biologically functionally equivalent thereto, anda pharmaceutically acceptable carrier.

[0042] Yet another object of the present invention is to provide amethod of immunizing poultry, comprising administering to said poultryany of the vaccines of the present invention.

[0043] Further scope of the applicability of the present invention willbecome apparent from the detailed description and drawings providedbelow. However, it should be understood that the detailed descriptionand specific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The above and other objects, features, and advantages of thepresent invention will be better understood from the following detaileddescriptions taken in conjunction with the accompanying drawings, all ofwhich are given by way of illustration only, and are not limitative ofthe present invention, in which:

[0045]FIG. 1 shows the location of the UL32 gene homologue in the genomeof MDV. The upper part of the figure is a schematic representation ofthe MDV genome. Abbreviations: TRL:terminal repeat adjacent to uniquelong region; UL: unique long region; IRL: internal repeat adjacent tounique long region; IRS: internal repeat adjacent to unique shortregion; US: unique short region; TRS: terminal repeat adjacent to uniqueshort region. The middle part of the figure shows the correspondingBamHI map of the MDV genome. The lower part of the figure shows detailsof the BamHI-E fragment containing the UL30, UL31, UL33, and UL34homologues. Arrows indicate the locatins of the five genes, and point intheir transcriptional directions, respectively.

[0046]FIG. 2 shows the DNA sequence of UL32 gene (SEQ ID NO:1).

[0047]FIG. 3 shows the amino acid sequence of the protein encoded by theUL32 gene (SEQ ID NO:2).

[0048]FIG. 4 shows the construction of plasmid vector pGTPs.

[0049] FIGS. 5A-5D show the construction of transfer vectorspNZ29RMDUL32 and pNZ29RMDgBUL32.

[0050]FIG. 6 shows immunoprecipitation using anti-trpE-UL32 fusionprotein antibody. Lane 1: uninfected CEF cells; lane 2: CEF cellsinfected with MDV-1 (GA strain); lane 3: CEF cells infected with MDV-2(SB-1 strain;) lane 4: CEF cells infected with HVT (FC126 strain).

[0051]FIG. 7 shows the mobility shift assay of immunoprecipitants aftertreatment with endoglycosidases. Lane 1: no treatment; lanes 2 and 3:O-glycanase; lane 4: endo-H; lane 5: PNGase.

[0052]FIG. 8 shows the immunoprecipitation of cells infected withrecFPV/MD-gB/UL32 or with the GA strain of MDV. Lane 1: CEF cellsinfected with recFPV/MD-gB/UL32 with monoclonal antibody 1AN86 specificfor MD gB; lane 2: uninfected CEF cells with monoclonal antibody 1AN86;lane 3: CEF cells infected with recFPV/MD-gB/UL32 with monoclonalantibody for gp82; lane 4: CEF cells infected with the GA strain of MDVwith monoclonal antibody for gp82.

[0053] FIGS. 9A-9B show the construction of the plasmids pNZ29RMDgE andpNZ29RMDgEgI.

[0054] FIGS. 10A-10 show the construction of the plasmids pNZ29RMDgBgE,pNZ29MDgBgEgI and pNZ29RMDgBgEgIUL32.

DETAILED DESCRIPTION OF THE INVENTION

[0055] The following detailed description of the invention is providedto aid those skilled in the art in practicing the present invention.Even so, the following detailed description of the invention should notbe construed to unduly limit the present invention, as modifications andvariations in the embodiments herein discussed may be made by those ofordinary skill in the art without departing from the spirit or scope ofthe present inventive discovery.

[0056] The contents of each of the references cited herein are hereinincorporated by reference in their entirety.

[0057] The UL32 gene and the polypeptide encoded thereby

[0058] The UL32 gene of the present invention is a 1926 base long DNAobtained from Marek's disease virus comprising the sequence fromnucleotides 1-1926 of SEQ ID NO:1, or a DNA sequence originating fromMDV substantially equal to the 1926 base long DNA that retains thefunctional activity thereof. Although the DNA sequence shown in SEQ IDNO:1 is that from obtained from strain GA, the present invention is notlimited to the gene originating from the GA strain alone.

[0059] Biologically Functionally Equivalent DNA Fragments

[0060] The nucleic acid sequences disclosed herein, or theirbiologically functional equivalents, can be used in accordance with thepresent invention. The phrase “biologically functional equivalents,” asused herein, denotes nucleic acid sequences exhibiting the same orsimilar biological activity/immunoprotective activity as the particularnucleic acid sequences described herein, i.e., when introduced intoviral hosts in a functionally operable manner so that they areexpressed, they elicit a protective immune response.

[0061] For example, the nucleic acid sequences described herein can bealtered by base substitutions, insertions additions, or deletions toproduce biologically functionally equivalent nucleic acids that encodeproteins conferring immunity to MDV in vivo. In addition, due to thedegeneracy of the genetic code, other DNA sequences that encodesubstantially the same amino acid sequences as described herein andconfer immunity Lo MDV in vivo caii be used in the practice of thepresent invention. These include, but are not limited to, nucleotidesequences comprising all or portions of the viral DNAs described hereinor the corresponding mRNAs or cDNAs that are altered by the substitutionof different codons that encode a physiologically functionallyequivalent amino acid residue within the protein sequence, thusproducing a silent change. Similarly, the proteins conferring immunityto MDV, or derivatives thereof, encoded by the present inventioninclude, but are not limited to, those containing all of the amino acidsequences encoded by the DNA sequences substantially as describedherein, including altered sequences in which functionally equivalentamino acid residues are substituted for residues within the sequence,resulting in a silent change. For example, one or more amino acidresidues within the sequence can be substituted with another amino acidof similar polarity which acts as a functional equivalent, resulting ina silent alteration. Substitutes for an amino acid within the sequencemay be selected from other members of the class to which the amino acidbelongs. For example, fungible nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine. Fungible polar neutral amino acids includeglycine, serine, thereonine, cysteine, tyrosine, asparagine, andglutamine. Fungible positively charged (basic) amino acids includearginine, lysine, and histidine. The negatively charged (acidic) aminoacids include aspartic acid and glutamic acid.

[0062] The variants of the genomic DNAs, the corresponding mRNAs orcDNAs, and proteins contemplated herein should possess more than 75%homology, preferably more than 85% homology, and most preferably morethan 95% homology, to the naturally occurring viral DNAs, thecorresponding mRNAs or cDNAs, and proteins discussed herein.

[0063] Also included within the scope of the present invention are gp82protein fragments, which are the expression products of the UL32 gene,or derivatives thereof that are differentially modified during or aftertranslation, e.g., by glycosylation, proteolytic cleavage, etc.

[0064] In the present invention, DNAs substantially biologicallyfunctionally equivalent to the UL32 gene possessing the nucleotidesequence 1-1926 of the nucleotide sequence shown in SEQ ID NO:1 aredefined either as DNAs originating from MDV, the length of which hasbeen altered by either natural or artificial mutations such as partialnucleotide deletion, insertion, addition, or the like, so that when theentire length of SEQ ID NO:1 is 100%, the resulting sequence has anapproximate length of 60-120% of that of SEQ ID NO:1, preferably80-110%, or a gene partially (herein partially means usually 20% orless, preferably 10% or less, and more preferably 5% or less of theentire length) replaced and altered by either natural or artificialmutations so that the nucleotide sequence codes for different aminoacids, but wherein the resulting protein retains the immunoprotectiveeffect of the naturally occurring protein. The mutated DNA created inthis manner must usually encode a protein having 75% or greater,preferably 85% or greater, and more preferably 95% or greater,similarity to the amino acid sequence of the UL32 protein (SEQ ID NO:2)encoded by the nucleotide sequence of SEQ ID NO:1.

[0065] In the present invention, similarity means that which is measuredby the method of Gonnet et al. (Science 256:1443-1445 (1992)).

[0066] In the present invention, the methods employed to createartificial mutations are not specifically limited, and therefore suchmutations can be produced by any of the standard means. For example, theUL32 gene obtained from a field strain MDV can be reinserted aftertreatment with appropriate restriction enzymes and insertion (ordeletion) of appropriate DNA fragments so that the proper amino acidreading frame is preserved. In addition, the in vitro mutagenesismethods described by Frits Eckstein et al., (Nucleic Acid Research10:6487-6497 (1982)), Osuna et al. (Critical Reviews in Microbiology20:107-116 (1994), and other methods can be used to alter a nucleotidesequence so that part of the amino acid sequence is translated intodifferent amino acids. Biologically functional equivalents to thenucleic acid fragments disclosed herein can be selected for using thetechniques described in Examples 3-8, below.

[0067] The DNAs described above that have been mutated by theabove-mentioned methods and other methods are considered to possessbiological function substantially equal to that of the UL32 gene of thepresent invention. The UL32 gene of the present invention can functionto suppress symptoms of MDV infection in chickens when inoculated as arecombinant avipoxvirus carrying the UL32 gene. If the effect of aparticular mutated DNA differs from the effect of the UL32 gene derivedfrom MDV strain GA ±20% or less, such DNA is considered to possess afunction substantially equal to that of the UL32 gene of the presentinvention.

[0068] The aforementioned gene of the present invention is presumablyconsidered to exist in the genome of all MDV serotypes. Therefore, thegene of the present invention can be obtained from any MDV strain byconventional methods, for example Southern hybridization using probesarbitrarily selected from the nucleotide sequence shown in SEQ ID NO:1.

[0069] Sources of the UL32 gene include the serotype 1 strains such asGA, RB1B, CVI988, 584A and Md5; serotype 2 strains such as SB-1 and301B/l; and serotype 3 strains such as FC126, YT-7 and H2. Among these,serotype 1 is preferable.

[0070] The polypeptides of the present invention possess the amino acidsequence encoded by the aforementioned DNA sequences, and suchpolypeptides can be easily produced and purified by the usual methodsusing recombinant viruses as described below.

[0071] Recombinant Viruses

[0072] The recombinant viruses of the present invention harbor the UL32gene of the present invention. In addition, they may also harbor antigengenes other than the UL32 gene. An example of the preparation of suchrecombinant viruses is as follows.

[0073] A plasmid which contains a nonessential region of the parentvirus is constructed. The UL32 gene is placed under control of apromoter that functions in the parent virus. A plasmid vector isobtained by inserting the UL32 construct into the nonessential region ofthe above plasmid. In the next step, homologous recombination is inducedby introducing the plasmid vector into cells infected with the parentvirus. Recombinant viruses are obtained by subjecting the resultingviruses to selection and purification.

[0074] Parent virus

[0075] In the present invention, “parent virus” means a virus in whichthe UL32 gene and other genes can be inserted. This can be any kind ofvirus. For example, for use as a vaccine, a virus that can infect thebirds to be vaccinated is preferable. Examples of such viruses includeviruses generally used for recombination: poxviruses, includingorthopoxviruses such as racoon poxvirus and vaccinia virus, andavipoxviruses (APV) such as pigeon poxvirus, fowl poxvirus, canarypoxvirus, quail poxvirus, and turkey poxvirus; herpesviruses such asturkey herpesvirus (HVT) and infectious laryngotracheitis virus (ILTV);adenoviruses; and influenza viruses.

[0076] Vaccinia viruses include the Copenhagen strain and the WR strain.Avipoxviruses include viruses that can replicate in poultry cells suchas those of chicken, turkey, and duck, for example, FPV, pigeonpoxvirus, canary poxvirus, turkey poxvirus, and quail poxvirus. FPVstrains include ATCC VR-251, ATCC VR-250, ATCC VR-229, ATCC VR-249, ATCCVR-288, Nishigahara, Shisui, and the CEVA vaccine strain. Among virusesderived from the CEVA vaccine strain, there are strains that form largeplaques upon infection of chick embryo fibroblast cells (CEF), andtherefore belong to the narrow definition of FPV. Other viruses like theNP strain (derived from the Nakano strain of chicken-fetus-adaptedpigeon poxvirus) are closely related FPV of the narrow definition, andare used to produce live chicken poxvirus vaccines. Herpesvirusesinclude HVT such as the FC126 and H2 strains, which can be obtained fromNippon Institute for Biological Science (Nisseiken Co., Ltd.), and theYT-7 strain, which can be obtained from the Chemo-Sero-TherapeuticResearch Institute (Kagaku Oyobi Kesseiryoho Kenkyusho); ILTV such asstrains like C7 and CE, which are currently used as anti-ILTV vaccines,and the NS-175 strain.

[0077] Among these viruses, avipoxviruses and avian herpesviruses areespecially preferable.

[0078] Nonessential regions

[0079] The nonessential region used in the present invention is definedas a region that is not essential for replication of the parent virus.For example, if the parent virus is a vaccinia virus, the TK gene regionand the HA gene region are nonessential regions. If the parent virus isan avipoxvirus, the TK gene region of turkeypox, fowlpox, pigeonpox, andquailpox viruses and the region described in Japanese Patent ApplicationLaid-open (Kokai) No. 168279/1989 can be used. In addition, the regionswhich homologously recombine with these regions are examples ofnonessential regions. The DNA fragments of the APV-NP strain aredescribed in the above Japanese Patent Application Laid-open (Kokai) No.168279/1989 (EcoRI fragment (7.3 kbp), EcoRI-HindIII fragment(approximately 5.0 kbp), BamHI fragment (approximately 4.0 kbp), andHindIII fragment (approximately 5.2 kbp). If the parent virus is aherpesvirus, nonessential regions include the TK gene region, the regionhomologous to the gC gene of herpes simplex virus (gC homologue), andthe unique short regions such as the Us2 homologue of herpes simplexvirus.

[0080] Vectors containing nonessential regions

[0081] Vectors containing nonessential regions are used in theconstruction of vectors for recombination described below. Such vectorscan be constructed by the usual methods, for example, by inserting thenonessential regions described above into vectors treated withappropriate restriction enzymes. Vectors which can be used in theconstruction include plasmids such as pBR322, pBR325, pUC7, pUC8, pUC18,and the like; phages such as λ and M13, and the like; and cosmids suchas pHC79 and the like.

[0082] Transfer vectors for recombination

[0083] Transfer vectors for recombination used in the present inventioncontain nonessential regions into which is inserted the UL32 gene, otherantigen genes which may be optionally employed, and promoters whichcontrol these genes. Such vectors can be constructed by inserting intothe viral nonessential regions of the above-mentioned vectors containingnonessential regions the UL32 gene of the present invention as well asantigen genes selected as needed with promoters which control thesegenes at the 5′ upstream position of each gene.

[0084] Furthermore, in order to facilitate efficient purification ofrecombinant viruses, marker genes such as the E. coli lacZ gene may beinserted with promoters described below which control such genes.

[0085] Antigen genes

[0086] In the present invention, an antigen gene or genes in addition tothe UL32 gene can be employed. Such antigen genes are defined as genesthat encode antigen proteins expressed by poultry pathogens (bacteria,viruses, etc.), and that can also be expressed as antigenic proteins bythe process of transcription and translation when inserted into a parentvirus Preferable examples of antigen-protein encoding DNAs (antigengenes) include the genes encoding the MDV glycoproteins gB, gI, gE, etc.(gB: Ross et al. J. Gen Virol. 70:1989-1804 (1988); gI and gE: Veliceret al., (U.S. Pat. No. 5,252,716); the gene encoding NDV HN (Miller etal. J. Gen. Virol. 67:1917-1927 (1986)), the gene encoding the F protein(McGinnes et al. Virus Res. 5:343-356 (1986)), the gene encoding thestructural protein VP2 of infectious bursal disease virus (Bayliss etal. J. Gen. Virol. 71:1303-1312 (1990)), and other genes encodingantigens involved in protection against infection.

[0087] Any gE gene or gI gene is usable in the present invention so longas the gE gene or gI gene is derived from the genome of a Marek'sdisease virus.

[0088] Said gE gene or said gI gene is not necessarily a gene having thecomplete sequence of a gE or gI gene. If a gene encodes a protein havingsubstantially the same function as a protein encoded by a gE or gI gene,such a gene is also usable in the present invention.

[0089] In the present invention, the phrase “substantially biologicallyfunctionally equivalent gene to said gE gene or said gI gene” denotes agene that has an approximate length of about 80-120%, preferably about90-110%, of the length of said gE gene or said gI gene. The length isaltered by either natural or artificial mutations such as nucleotidedeletions, insertions, addition of new sequence, or the like, whereinthe entire length of said gE gene or said gI gene is taken as 100%. Asubstantially biologically functionally equivalent gene is also a genepartially replaced and altered by either natural or artificial mutationsso that the nucleotide sequence codes for different amino acids, butwherein the resulting protein retains the immunoprotective effect of thenaturally occurring protein. Herein “partially” means 20% or less,preferably 10% or less, and more preferably 5% or less of the entiresequence of the gene is altered. The mutated DNA created in this mannermust usually encode a protein having 80% or greater, preferably 90% ormore, and more preferably 95% or greater, homology to the amino sequenceof the protein naturally encoded by said gE or said gI gene. In thisrespect, the term “homology” means identity as measured using DNAsequence analysis software, such as DNASIS, available from Takara ShuzoKK.

[0090] In the present invention, the methods employed to createartificial mutations are not specifically limited, and therefore suchmutations can be produced by any of the standard means. For example, thenaturally occurring gE gene obtained from a field strain of MDV can bereinserted into a vector after treatment with appropriate restrictionenzymes and insertion (or deletion) of appropriate DNA fragments so thatthe proper amino acid reading frame is preserved. In addition, the invitro mutagenesis methods described by Frits Eckstein et al., (NucleicAcid Research 10:6487-6497 (1982)), and other methods can be used toalter a nucleotide sequence so that part of the amino acid sequence istranslated into amino acids different from the amino acids normallyfound in the protein.

[0091] The proteins encoded by the DNAs described above that have beenmutated by the above-mentioned methods and other methods are consideredto possess biological function substantially equal to that of theprotein encoded by the naturally occurring gE or gI. The phrase “thefunction of the naturally occurring gE or gI” means a function ofinhibiting the immune response derived from complement-dependent orantibody-dependent cell damage as a corollary of having a physiologicalactivity that a conjugate gI-gE or gE itself bonds to F_(c) portion ofIgG. The phrase “function substantially equal to that of the naturallyoccurring gE or gI” means an activity equal to about 1.5 times or more,preferably about 2.0 times or more of the protection effect observed inthe group of animals vaccinated with a recombinant avipox virus intowhich an antigen gene such as a UL32 gene, a gE gene and, optionally, agI gene have been incorporated without making gE and gI expressed. Theactivity is determined by using animals retaining about 50% of thetransfer antibody just after the birth with said recombinant virus. Thevaccinated animals are then infected with a virulent strain of MDV whenthe transfer antibody has become substantially lost (to about 10% orless of the level found in the newborn animals). “Transfer antibody” isantibody passed from the mother to the newborn during egg development.

[0092] As an exemplified gene for said gE derived from Marek's diseasevirus or a gene substantially equalivalent thereto, one may give a DNAencoding the amino acid sequence derived from Marek's disease virus typeI strain GA (SEQ ID NO:29). As an example of said gI gene or anequivalent thereof one may give a DNA encoding the amino acid sequencederived from Marek's disease virus type I strain GA (SEQ ID NO:28).

[0093] In the present invention, the gE gene can provide an effect as avaccine which is seldom affected by the transfer antibody even if the gEgene is used singly. However, more effective protection can be attainedif a recombinant virus into which the gI gene has been incorporatedtogether with the gE gene is used.

[0094] There are no specific limitations with respect to the order ofthe linkage between the gE gene, the gI and an antigen gene which willbe described hereinafter in detail as far as respective genes are linkedin such a manner that each of said gE, said gI and said antigen gene aresubstantially expressed. That is, a possible linkage would be gE gene—GIgene—antigen gene; gI gene—gE gene—antigen gene; gE gene—antigen gene—gIgene; gI gene—antigen gene—gE gene; antigen gene —gE gene—gI gene orantigen gene—gI gene—gE gene; ordered from the 5′ end. Alternatively thegE gene can be linked after the gI gene, that is, at the 3′-position andthe antigen gene can be linked to the resultant gI-gE construct at the3′- or 5′-position.

[0095] There are no specific limitations as to the position for linkinga useful marker gene.

[0096] Neither are there specific limitations as to the methods forlinking those genes and any conventional method such as the one in whicha suitable linker is employed for the linkage or the one in which arecombinant vector is directly produced by homologous recombinationusing respective gene-containing vectors.

[0097] Promoters

[0098] The promoters used in the present invention are not limited toparticular promoters, and can be any promoters as far as they exhibitpromoter activity in hosts infected with recombinant viruses. They maybe natural virus promoters, modified natural virus promoters, orsynthetic promoters.

[0099] Natural virus promoters include, in cases wherein the parentvirus is a poxvirus such as VV, APV, and the like, the promoter of thevaccinia virus gene that encodes the 7.5 KDa polypeptide, the promoterof the vaccinia virus gene that encodes the 11 KDa polypeptide, and thepromoter of the vaccinia virus gene that encodes thymidine kinase. Thesepromoters can be modified by alteration, addition, deletion, and gain orloss of nucleotides so long as they exhibit promoter activity.

[0100] Examples of synthetic promoters include the synthetic promoterwhich contains both early and late promoter sequences (A. J. Davidson etal. J. Mol. Biol. 215:749-769 and 771-781 (1989)), and derivativesthereof that have been partially modified by deletion and/or alterationof nucleotides, but still retain their promoter activity. An example isa sequence having the nucleotide sequence:

[0101] 5′-TTTTTTTTTTTTTTTTTGGCATATAAATAATAAATACAATAATTAATTACGCGTAAAAATTGAAAAACTATTCTAATTTATTGCACTC-3′ (SEQ ID NO:3). This syntheticpromoter and its modified forms contain a long stretch of T bases attheir 5′ end. It has been determined that this all T region shouldpreferably contain 15-40, more preferably 18-30, T bases for promoteractivity and expression of antigen genes.

[0102] It is possible to insert promoters in a manner such that each ofthe UL32, additional antigen, and marker genes is controlledindividually. In such a construct, the promoters connected to the genesneed not be the same promoters.

[0103] Methods of constructing recombinant viruses

[0104] There are no specific limitations with respect to the method forconstructing recombinant viruses. Such constructs can be produced byconventional methods. For example, recombinant viruses can be obtainedthrough induction of homologous recombination between a vector and thevirus genome present in infected cells by introducing a recombinantvector containing the UL32, additional antigen, and other genes intocells that have been infected with a parent virus. Recombinant virusesobtained in this manner can be purified by infecting host cells culturedin a medium such as Eagle's MEM, and by selecting candidate strains bythe use of the hybridization method with the inserted antigen gene as aprobe and by expression of the marker gene inserted with the antigengene. Purified candidate strains thus obtained can be confirmed asdesired recombinant viruses by methods such as immunoassays using anantibody against the polypeptide encoded by the inserted antigen gene.For example, APV containing the lacZ gene as the marker gene expressesβ-galactosidase, and therefore forms blue plaques in the presence of oneof its substrates, Bluogal (manufactured by GIBCO-BRL), thus enablingselection and purification.

[0105] Host cells are not limited to particular cells, and can be anycells which the virus in use can infect and replicate in, for example,chicken embryo fibroblast (CEF) cells and chicken embryo chorioallantoicmembrane cells in the case of FPV.

[0106] Poultry vaccines

[0107] The vaccines of the present invention include vaccines containingas an active ingredient the UL32 gene, or a recombinant vector orvectors which contain the UL32 gene (vaccine I), vaccines containing asan active ingredient recombinant virus or viruses that contain the UL32gene as well as other antigen genes inserted as needed (vaccine II),vaccines containing other vaccine ingredients in addition to vaccine II(vaccine III), and vaccines containing as an active ingredient thepolypeptide encoded by the UL32 gene (vaccine IV).

[0108] Vaccines of the present invention are administered in effectiveamounts as described below.

[0109] Vaccines containing as an active ingredient the UL32 gene, or arecombinant vector or vectors which contain the UL32 gene (vaccine I)

[0110] Recombinant vectors used herein can be any vectors that containthe UL32 gene of the present invention with an inserted promotersequence that is functional in eukaryotic cells, but preferably plasmidvectors that can be easily propagated in E. coli. The promoterfunctional in eukaryotic cells can be of either cellular origin, such asthe actin gene promoter, or viral origin, such as a cytomegaloviruspromoter or a retrovirus LTR.

[0111] Vaccines in the form of purified DNA can be administered byintramuscular, intravenous, intraperitoneal, or subcutaneous injection,but intramuscular injection is most preferred. Direct DNA administrationcan be accomplished with a syringe, but more preferably by a so-calledGene-gun. DNA should be administered in a dose that can achievesufficient immunological induction, usually 10-300 μg divided into 2 to4 inoculations. If a Gene-gun is used, the DNA dosage can be reduced toone-tenth the above dose or less.

[0112] Vaccines containing as an active ingredient recombinant virus orviruses which contain the UL32 gene as well as other antigen genesinserted as needed (vaccine II)

[0113] Vaccine II is composed of one or more recombinant virusesselected from (i) a recombinant virus carrying the UL32 gene of thepresent invention, (ii) a recombinant virus carrying the UL32 gene aswell as another antigen gene(s), and (iii) a mixture thereof. Therecombinant virus (ii) is superior to the recombinant virus (i). Saidrecombinant virus can be used singly or in combination with 2-3 otherrecombinant viruses of the present invention. Also, in addition torecombinant viruses, pharmacologically inactive materials such asphysiological saline and stabilizing agents can be added.

[0114] There are no specific limitations on the method for preparingvaccine II of the present invention. For example, cells susceptible torecombinant viruses of the present invention can be infected with one ofthe recombinant viruses of the present invention and cultured until therecombinant virus propagates in these cells. Cells are then collectedand disrupted. Centrifugation of the disrupted cells separates the hightiter supernatant from the precipitate. The supernatant which isessentially free of host cells and contains cell culture medium and therecombinant virus can be used as a vaccine of the present invention. Thevaccine can be diluted prior to use with a pharmacologically acceptablediluent such as physiological saline. The supernatant can also be usedas a freeze-dried vaccine after lyophilization.

[0115] If one of the vaccines of the present invention is to be used asa vaccine for chickens, administration thereof can be achieved by anymethod by which the recombinant virus in the vaccine can infect poultryand induce protective immunity in the infected birds. For example,inoculation can be performed by stabbing the wing web or scratching theskin. Subcutaneous injection using a needle or other tools can also beused for inoculation. Oral administration can be achieved by suspendingthe vaccine in poultry drinking water or mixing the vaccine in solidfeed. In addition, among other methods, inhalation of the vaccine in theform of an aerosol or spray, intravenous injection, intramuscularinjection, and intraperitoneal injection can also be used.

[0116] The dosage for chickens is usually 10-10⁶ plaque forming units(PFU), preferably 10²-10⁵ PFU, per bird. For injection, vaccinescontaining the above titer should be diluted with a pharmacologicallyacceptable liquid such as physiological saline to a final volume ofapproximately 0.1 ml or 0.01 ml in the case of wing web administration.

[0117] The vaccines of the present invention can be stored and usedunder normal conditions. For example, lyophilized recombinant viruses ofthe present invention can be stored in a refrigerator (0-4° C.), or evenat room temperature (20-22° C.) for a short period. Virus suspensionscan be stored frozen at −20° C. to −70° C.

[0118] Vaccines containing other vaccine ingredients in addition tovaccine II (vaccine III)

[0119] Other vaccine ingredients in vaccine III include, for example,antigen genes, recombinant viruses which carry one or more antigen genesbut not the UL32 gene, inactivated vaccines, and component vaccines.Examples of other vaccine ingredients defined above include recombinantAPV which does not contain the UL32 gene, Marek's disease vaccines(FC126 strain of HVT, strain CVI988 of serotype 1 MDV, SB-1 and 301B/1strains of serotype 2 MDV), ILTV vaccines, and avian encephalomyelitisvaccines. Vaccine III can be produced by mixing these and vaccine IIdescribed above by a pharmacologically acceptable method. In fact,mixing with a commercially available anti-Marek's disease vaccine, HVTvaccine, proves to be highly effective.

[0120] In such cases, the dosage of each vaccine can be set arbitrarily.For example, the dosage for vaccine II is set at {fraction (1/10)} to 1times the dosage described above, i.e., for chickens, 10⁰ to 10⁶,preferably 10¹ to 10⁵, PFU per bird. Similarly, other vaccines shouldalso be used at about {fraction (1/10)} to 1 times the usual dosage.

[0121] For storage of vaccine III, the characteristics of eachingredient should be taken into consideration. All the ingredients canbe stored together or separately.

[0122] Also, it should be pointed out that the most distinctive featureof vaccine III is the use of vaccine II in combination with otheringredients. Therefore, simultaneous administration of vaccine II andother vaccine ingredients as well as administration of a mixture ofvaccine II and other ingredients is expected to produce excellenteffects. “Simultaneous” herein means within 7 days, preferably 3 days,before or after the administration of vaccine II.

[0123] Vaccines containing as an active ingredient the polypeptideencoded by the UL32 gene (vaccine IV)

[0124] The polypeptide encoded by the UL32 gene can be prepared byeither infecting susceptible host cells with the plasmid vectors orvirus vectors mentioned above in the descriptions of vaccines I and II,or by expressing the UL32 gene in expression systems such as E. coli,yeast, and baculovirus. The polypeptide can then be used as a vaccinewith a common adjuvant such as Alum and FCA. Of course,pharmacologically acceptable vehicles such as physiological saline andstabilizing agents can be added to the vaccine.

[0125] The present invention will be described in detail in thefollowing Examples. However, it should be understood that the Examplespresented below are merely illustrative, and not limiting of the presentinvention.

EXAMPLE 1 Cloning And Sequencing Of The UL32 gene

[0126] The BamHI-E fragment of MDV DNA in pACYC184 (Fukuchi et al. J.Virol. 51:102-109 (1984)) was cloned into the BamHI site of pUC18 forsequencing, and various subclones were prepared from this fragment usingappropriate restriction enzymes. The fragment was treated withexonuclease III followed by mung bean nuclease to make sets of deletionmutants when necessary. DNA sequencing was performed on double strandedplasmid by the dideoxy chain termination method using [α³⁵S] DATP (NEN)and the TAQuence version 2.0 DNA Sequencing Kit (United StatesBiochemical Corporation) as suggested by the manufacturer. The ORF ofUL32 has a size of 1,926 bps and is leftward, and contains an averagebase composition of 27.6% A, 24.2% G, 19.8% C, and 28.3% T.

[0127] The DNA sequence upstream and downstream of UL32 was analyzed forputative transcriptional control elements. A consensus “TATA” box(5′-TATTAA-3′), characteristic of many eucaroytic and also herpesviralpromoters, is located −151 nucleotides upstream from the proposedinitiation codon. The sequence 5′-CCGAATGG-3′, which resides 87nucleotides upstream from the “TATA” box, exhibits similarities to the“CAT” box consensus sequence (5′-GGYTCAATCT-3′) (SEQ ID NO:4). Apossible SP-1 binding element (CCGCCC) is located at position -337nucleotides upstream from the start codon.

[0128] Regarding 3′ elements of UL32, there is no suitable poly Asequence (AATAAA or ATTAAA) downstream from the ORF. However, thereexist a “CAT” box (5′-GACCAATCC-3′) and a “TATA” box (5′-TATAAA-3′) atthe C-terminal of UL32.

EXAMPLE 2 Identification of The UL32 Gene Product

[0129] The polypeptide predicted from the nucleotide sequence (SEQ IDNO:1 and FIG. 1) comprises 641 amino acids with a calculated molecularweight of 71.5 KDa. The amino acid sequence is shown in FIG. 2 and SEQID NO:2. The polypeptide is far from a typical classical membraneprotein. There is no signal sequence, and it has only 4 potentialdomains (amino acid residues 86-104; 124-140; 464-482; and 586-597) thatmay interact with or span the membrane. In this polypeptide, there aretwo potential N-linked glycosylation sites (amino acid resides 47 and242).

EXAMPLE 3 Construction of pGTPs, The Plasmid For Antigen Gene Insertion

[0130] The plasmid pGTPs is constructed as follows. A synthetic DNAhaving the sequence 5′-AGCTGCCCCCCCGGCAAGCTTGCA-3′ (SEQ ID NO:5) isinserted into the HindIII-PstI sites of pUC18. A synthetic DNA havingthe sequence 5′-TCGACATTTTTATGTAC-3′ (SEQ ID NO:6) is then inserted intothe SaII-KpnI sites, followed by insertion of the annealing productbetween the synthetic DNA having the sequence5′-AATTCGGCCGGGGGGGCCAGCT-3′ (SEQ ID NO:7) and a synthetic DNA havingthe sequence 5′-GGCCCCCCCGGCCG-3′ (SEQ ID NO:8) into the SacI-EcoRIsites. Finally, the 140 bp HindIII-SalI fragment from pNZ1729R (U.S.Pat. No. 5,369,025) is inserted into the HindIII-SalI site of theresulting plasmid (FIG. 4).

EXAMPLE 4 Construction Of Transfer Vectors PNZ29RMDUL32 andpNZ29RMDgBUL32

[0131] The polymerase chain reaction (PCR) was used to clone theMDV-UL32 gene and to remove the potential poxvirus early transcriptiontermination signals (Yuen et al. PNAS USA 84:6417-6421 (1987)) from thisgene. Three sets of primers were used: Set 1:5′-CCCCGGATCCGGCCATGGCCAACCGC-3′ (32-a) (SEQ ID NO:9) (BamHI siteunderlined) and 5′-AAGAATGCATAATCTGCCATCCAT-3′ (32-bR) (SEQ ID NO:10)(EcoT22I site underlined); Set 2: 5′-GATTATGCATTCTTATGTTCCAAATG-3′(32-b) (SEQ ID NO:11) (EcoT22I site underlined) and5′-ACAGCCATGGAGAAAGAAATGTCTCTGAATATC-3′ (32-cR) (SEQ ID NO:12) (Ncolsite underlined); Set 3: 5′-TTCTCCATGGCTGTTTTCGAACG-3′ (32-c) (SEQ IDNO:13) (NcoI site underlined) and 5′-CCCCGTCGACTTACACGTAGACTCCTAATG-3′(32 -dR) (SEQ ID NO:14) (SalI site underlined).

[0132] MDV genomic DNA to be used as PCR templates was prepared asfollows. CEF cells infected with the GA strain of MDV were recoveredfrom tissue culture dishes by trypsinization, washed twice with PBS, andsuspended in Proteinase K buffer (10 mM Tris-HCl, pH7.8, 5mM EDTA, 0.5%SDS). Proteinase K (Boehringer Mannheim) was then added to a finalconcentration of 50 μg/ml, followed by incubation at 55° C. for 2 hours.Proteins were removed by two phenol/chloroform extractions. Afteradddition of two volumes of ethanol followed by incubation at −20° C.for 20 minutes, genomic DNA of the GA strain of MDV was recovered bycentrifugation.

[0133] Primer 32-a contains the nucleotide sequence of nucleotides 1-12of SEQ ID NO:1, and has an upstream BamHI site for cloning. Primer 32-bRcontains the nucleotide sequence of nucleotides 381-358 (reverseorientation, complementary strand) of SEQ ID NO:1 with mutations atnucleotide 376 (T to A) and nucleotide 379 (T to C) of the sense strand.Though by introduction of these mutations the nucleotide sequence ofthis region (371-385) changes from TATGCTTTTTTATGT (SEQ ID NO:15) toTATGCATTCTTATGT (SEQ ID NO:16), there is no change in the amino acidsequence encoded by these sequences; they both code forTyr-Ala-Phe-Leu-Cys (SEQ ID NO:17). The mutation at nucleotide 376 wasintroduced to create a cutting site for the restriction enzyme EcoT22I.The mutation at nucleotide 379 was introduced to remove the potentialpoxvirus early transcription termination signal (TTTTTNT; N being anarbitrary nucleotide).

[0134] Primer 32-b contains the nucleotide sequence of nucleotides368-393 of SEQ ID NO:1, with mutations at nucleotide 376 (T to A) andnucleotide 379 (T to C) of the sense strand. Though by introduction ofthese mutations the nucleotide sequence of this region (371-385) changesfrom TATGCTTTTTTATGT (SEQ ID NO:18) to TATGCATTCTTATGT (SEQ ID NO:16),there is no change in the amino acid sequence encoded by thesesequences; they both code for Tyr-Ala-Phe-Leu-Cys (SEQ ID NO:17). Themutation at nucleotide 376 was introduced to create a cutting site forthe restriction enzyme EcoT22I. The mutation at nucleotide 379 wasintroduced to remove the potential poxvirus early transcriptiontermination signal (TTTTTNT; N being an arbitrary nucleotide).

[0135] Primer 32-cR contains the nucleotide sequence of nucleotides529-561 (reverse orientation, complementary strand) of SEQ ID NO:1, withmutations at nucleotide 546 (T to C) and nucleotide 552 (G to C) of thesense strand. Though by introduction of these mutations the nucleotidesequence of this region (541-558) changes from CATTTTTTTCTGCATGGC (SEQID NO:19) to CATTTCTTTCTCCATGGC (SEQ ID NO20 ), there is no change inthe amino acid sequence encoded by these sequences; they both code forHis-Phe-Phe-Leu-His-Gly (SEQ ID NO:21). The mutation at nucleotide 546was introduced to remove the potential poxvirus early transcriptiontermination signal (TTTTTNT; N being an arbitrary nucleotide). Themutation at nucleotide 552 was introduced to created a cutting site forthe restriction enzyme NcoI.

[0136] Primer 32-c contains the nucleotide sequence of nucleotides548-570 of SEQ ID NO:i, with a mutation at nucleotide 552 (G to C) ofthe sense strand. Though by introduction of this mutation the nucleotidesequence of this region (541-558) changes from CATTTTTTTCTGCATGGC (SEQID NO:19) to CATTTCTTTCTCCATGGC (SEQ ID NO:20), there is no change inthe amino acid sequence encoded by these sequence; they both code forHis-Phe-Phe-Leu-His-Gly (SEQ ID NO:21). The mutation at nucleotide 552was introduced to create a cutting site for the restriction enzyme NcoI.

[0137] Primer 32-dR contains the nucleotide sequence of nucleotides1926-1907 (reverse orientation, complementary strand) of SEQ ID NO:1,and has an upstream Sall site for cloning.

[0138] Amplified fragments were cloned into the pGEM-T vector (PromegaCorp., Madison, Wis.) and analyzed by DNA sequencing. Each of threeplasmids, pGEM32ab, pGEM32bc, and pGEM32cd has an insert of the first,the second, and the third PCR fragment, respectively.

[0139] The 379 bp BamHI-EcoT22I fragment from pGEM32ab, the 180 bpEcoT22I-NcoI fragment from pGEM323bc, and the 1374 bp NcoI-SalI fragmentfrom pGEM32cd were cloned into BamHI/SalI-digested pGTPs. The resultingplasmid was named pGTPsUL32. pNZ1829R was derived from pNZ1729R(Yanagida et al. J. of Virolocy 66:1402-1408 (1992)) and annealingoligos having the sequence 5′-GGCCCCCCCGGCCG-3′ (SEQ ID NO.22) and5′-AATTCGGCCGqqqqqGCCAGCT-3′ (SEQ ID NO:23 ) between SacI and EcoRIsites located at the junction region of the lacZ gene and FPV DNA. A1933 bp BamHI-SalI fragment from pGTPsUL32 was cloned into BamHI/SalIdigested pNZ1829R to produce transfer vector pNZ29RMDUL32. PlasmidpNZ29RMDgBSfi was derived from pNZ29RMDgB-S (Yanagida et al. J. ofVirology 66:1402-1408 (1992)) with annealing oligos having the sequences5′-GGCCCCCCCGGCCG-3′ (SEQ ID NO:22) and 5′-AATTCGGCCGGGGGGGCCAGCT-3′(SEQ ID NO:2₃) between SacI and EcoRI sites located at the junctionregion of the lacZ gene and FPV DNA. A 2066 bp BgII fragment frompGTPsUL32 was cloned into Sf il digested pNZ29RMDgBSfi to obtaintransfer vector pNZ29RMDgBUL32.

EXAMPLE 5 Generation And Purification Of Recombinant FPVs

[0140] Procedures for transfection of FPV-infected cells with thetransfer vectors (pNZ29RMDUL32 or pNZ29RMDgBUL32 of Example 4) byelectroporation and generation of recombinants have been describedpreviously (Ogawa et al. Vaccine 8:486-490 (1990)). Approximately 3×10⁷CEF cells previously infected with FPV at a multiplicity of infection of0.1 were transfected with 10 μg of transfer vector. After 3 days ofincubation, progeny FPV were assayed for expression of lacZ in thepresence of Bluo-gal (600 μg/ml) in the agar overlay. Blue plaques wereremoved from the agar and clone purified until all FPV plaques wereblue.

[0141] The purified recombinant viruses were named recFPV/MD-UL32 orrecFPV/MD-gB/UL32.

EXAMPLE 6 Expression Of MDV gB And UL32 Antigens In Cell Culture

[0142] To test whether UL32 is translated in MDV-infected cells, CEFcells infected with GA strain, SB-1 strain, and HVT were radiolabeled,and immunoprecipitated with rabbit antiserum against the fusion proteintrpE-UL32. The immunoprecipitated samples were analyzed by 8% SDS-PAGEfollowed by fluorography (FIG. 6). Anti-trpE-UL32 fusion proteinantibody immunoprecipitated a protein with Mr 82,000 Da from the lysatesof MDV-1 (GA strain) and HVT-infected cells, respectively (Lanes 2 and4), but none from the lysates of MDV-2 (SB-1 strain) infected cells andCEF cells (Lane 3 and Lane 1). The apparent molecular weight is higherthan that calculated according to the deduced amino acid sequence. Toexamine the type of carbohydrate modification in this glycoprotein,immunoprecipitated proteins were treated with endoglycosidases. AfterO-glycanase treatment, a mobility shift from Mr 82,000 to Mr 76,000 wasobserved (FIG. 7, Lanes 2 and 3). No mobility shift was observed whenthe immunoprecipitated protein was treated with endo-H and PNGase (FIG.7, Lanes 4 and 5). Although there are two potential N-linkedglycosylation sites in the amino acid sequence of UL32, they are notlikely used. The size of the protein after O-glycanase treatment iscloser to that calculated from the deduced amino acid sequence.

[0143] In order to show that recFPV/MD-gB/UL32 synthesizes both the gBantigen and the UL32 antigen, CEF cultures infected with this virus wereexamined by IF using antibodies specifically raised against theseantigens. CEF cultures infected with recFPV/MD-gB/UL32 were incubated at37° C. until typical FPV plaques developed. These cultures were fixed incold acetone, and then reacted with appropriate dilutions of monoclonalantibody specific to MDV gB antigen (Silva et al. Viroloy 136:307-320(1984)) or a monoclonal antibody specific to MDV UL32 antigen. Thesecultures were then reacted with fluorescein-conjugated anti-mouseimmunoglobulins, and after thorough washing to remove non-specificstaining, they were examined microscopically under ultraviolet (UV)illumination. CEF cultures infected with non-recombinant parental FPVwere similarly stained. Specific cytoplasmic and membrane staining ofcells was observed in cultures infected with the recFPV/MD-gB/UL32 witheach of the monoclonal antibodies and not in cultures infected with thenon-recombinant parental FPV. These observations clearly show that therecombinant virus is capable of synthesizing the products of the gB andUL32 genes of MDV in cell cultures.

[0144] Primary CEF cultures infected with either parental or recombinantFPV at an moi of 5 were incubated at 37° C. for 4 hours. Then, themedium was replaced with 1 ml of fresh methionine-free medium andincubated for another hour. About 40 μCi of ³⁵S-methionine (NEN,Wilmington, Del.) were then added, and the cultures were incubated foran additional 5 hours. Cells were washed twice in PBS, scraped, andtransferred to a 15 ml Falcon tube. Cells were centrifuged, resuspendedin lysis buffer (150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100,0.1% SDS, and 10 mM Tris HCl, pH 7.5) and incubated at room temperaturefor 30 minutes. One half volume of 10% (v/v) S. aureus Cowan 1 (SAC) wasadded to the cell lysate, and incubated for 30 minutes on ice. Thelysate was then centrifuged, and the supernatant was collected. About 3μl of monoclonal antibody 1AN86 against MD gB (Silva et al. Virology136:307-320 (1984)) were added to 100 μl of lysate and incubated for 30minutes on ice. An equal volume of 10% (v/v) SAC was added and incubatedon ice for 30 minutes. Immunoprecipitates were then washed, suspended insample buffer, and then boiled. After centrifugation, the supernatantwas analyzed by sodium dodecylsulphate-polyacrylamide gelelectrophoresis (Laemmli Nature 207:680-685 (1970). FIG. 8 shows theresults of immunoprecipitation with monoclonal antibodies specific forMD gB and gp82 proteins. Lane 1: CEF cells infected withrecFPV/MD-gB/UL32 with monoclonal antibody for gB; Lane 2: CEF cellsinfected with wild-type FPV with monoclonal antibody for gB; Lane 3: CEFcells infected with recFPV/MD-gB/UL32 with monoclonal antibody for gp82;Lane 4: CEF cells infected with the GA strain of MDV with monoclonalantibody for gp82. Three identical bands of 100 kd, 60 kd, and 49 kd inmolecular weight were observed in extracts of cells infected withrecFPV/MD-gB/UL32 (FIG. 8, lane 1). A single band of 82 kd was observedusing Mab against gp82 in the extract of double recombinants. Theseresults demonstrate that recFPV/MD-gB/UL32 expresses both gB antigencomplex and UL 32 antigen.

EXAMPLE 7 Protection Of Antibody-Negative Chickens With Recombinant FPVsExpressing The gB Or UL32 Gene, Or Both, Against A Very Virulent StrainOf MDV (Md5)

[0145] Separate groups of 1-day-old chickens from 15×7 AB(−) chickenline susceptible to MD were vaccinated with 10⁵ plaque forming units(PFU) of recFPV/MD-gB (U.S. Pat. No. 5,369,025; Example 4), 10⁵ PFU ofrecFPV/MD-UL32 (Example 5), 10⁵ PFU of recFPV/MD-gB/UL32 (Example 5), or2×10³ PFU of the FC126 strain of HVT. Another group of similar chickenswas kept unvaccinated. All were kept in strict isolation. At 6 days ofage, all chickens were challenged with 5×10² PFU of the very virulentMd5 strain of MDV (Witter et al. Avian Dis. 24:210-232 (1980)). A fifthgroup of chickens was neither vaccinated nor challenged. Mortalitycaused by MD was recorded during the trial, and at the end of the 8 weektrial, all chickens were examined for gross lesions and tumors typicalof MD. The results of this study are presented in Table 1.

Table 1

[0146] Vaccination trial and evaluation of synergistic effects ofrecombinant FPV expressing the gB and UL32 genes of MDV in comparisonwith a conventional vaccine (FC126 of HVT) in Ab(−) chickens challengedwith the Md5 isolate of MDV at 5 days post vaccination MD % Vaccine No.tested Death lesion protection recFPV/MD-gB 14 0 4 71 recFPV/MD-gB/UL3214 0 0 100 recFPV/MD-UL32 14 2 12 14 HVT 14 0 3 79 none 14 11 14 0none-no challenge 14 0 0

[0147] A significant number of unvaccinated chickens died of MD beforethe end of the trial. On the other hand, chickens vaccinated withrecFPV/MD-gB, recFPV/MD-gB/UL32, or HVT were fully protected from deathdue to MD. The majority of chickens vaccinated with recFPV/MD-UL32 wereprotected from death due to MD, although they were not significantlyprotected from MD lesions. Seventy-one percent of chickens vaccinatedwith recFPV/MD-gB were similarly protected from MD lesions as comparedto 79% of those vaccinated with HVT. Those vaccinated withrecFPV/MD-gB/UL32 were fully protected (100%) against the very virulentMd5 strain of MDV, and showed no mortality and no lesions typical of MD.recFPV/MD-UL32 confers only 14% protection, but the death count is low,i.e., only 2 chickens. The protective effect of recFPV/MD-gB/UL32 is notmerely the sum of the effects of recFPV/MD-gB+recFPV/MD-UL32. Rather,the UL32 gene unexpectedly enhances the effect of the gB antigen gene.

EXAMPLE 8

[0148] Protection Of Antibody-Positive Chickens With Recombinant FPVsExpressing The gB Or The UL32 Gene, Or Both, Against A Very VirulentStrain Of MDV (Md5)

[0149] Two sets of experiments were performed. Each set was performed asfollows. Separate groups of 1-day-old chickens from 15×7 Ab(+) chickenline susceptible to MD were vaccinated with 10⁵ plaque forming units(PFU) of recFPV/MD-gB, 10⁵ PFU of recFPV/MD-UL32, 10⁵ PFU ofrecFPV/MD-gB/UL32, 2×10³ PFU of HVT, 2×10³ PFU of CVI988/Rispens(Rispens), 10³ PFU of SB-1+10³ PFU of HVT, 10⁵ PFU of recFPV/MD-gB+2×10³PFU of HVT or 10⁵ PFU of recFPV/MD-gB/UL32+2×10³ PFU of HVT vaccines.Another group of similar chickens was kept unvaccinated. All were keptin strict isolation. At 6 days of age all were challenged with 5×10² PFUof the very virulent Md5 strain of MDV. A tenth group of chickens wereneither vaccinated nor challenged. Mortality caused by MD was recordedduring the trial and at the end of the 8 week trial all chickens wereexamined for gross lesions and tumors typical of MD. The results of thisstudy are presented in Table 2. TABLE 2 Vaccination trial and evaluationof synergistic effects of recombinant FPV expressing the gB and UL32genes of MDV in comparison with monovalent and bivalent conventionalvaccines in Ab(+) chickens challenged with the Md5 isolate of MDV at 5days post vaccination Trial 1 Trial 2 Summary vaccine MD test % MD PI MDtest % MD PI MD test % MD PI recFPV/MD-gB 11 15 73 17 8 16 50 47 19 3161 33 recFPV/MD-UL32 10 14 71 19 13 17 76 19 23 31 74 18recFPV/MD-gB/UL32 3 17 18 80 5 17 29 69 8 34 24 74 HVT 5 15 33 62 11 1765 31 16 32 50 45 Rispens 6 17 35 60 8 16 50 47 14 33 42 53 SB1 + HVT 817 47 47 2 16 13 87 10 33 30 67 gB + HVT 4 17 24 73 3 17 18 81 7 34 2177 gB/UL32 + HVT 2 15 13 85 3 17 18 81 5 32 16 83 None 14 16 88 16 17 9430 33 91 None (no chal.) 0 17 0 0 17 0 0 34 0

[0150] Moderate levels of protection were observed in chickensvaccinated with recFPV/MD-gB, recFPV/MD-UL32 (U.S. Pat. No. 5,369,025example 4) (example 5) or HVT (cell associated FC126 strain of HVT).Whereas chickens vaccinated with recFPV/MD-gB/UL32 were significantlywell protected (74% on average protection index (PI={% MD in control-(%MD in test)/(% MD in control)}×100, which was even better than widelyused commercial bivalent SB-1 (another Serotype 2 vaccine (Witter etal., Avian Dis., 31 829-844 (1987))+HVT (67%) or CVI988/Rispens (53%),which is considered to be the best monovalent MDV vaccine in commercialuse in Europe.

[0151] Although either recFPV/MD-gB or HVT alone induced protection of33% and 45%, respectively, a combination of these two vaccines showed asignificant synergism of protection (77%).

EXAMPLE 9 Protection and Synergism by Recombinant Fowlpox VaccinesExpressing Genes from Marek's Disease Virus

[0152] Overview

[0153] Recombinant fowlpox viruses (recFPV) were constructed to expressgenes from serotype 1 Marek's disease virus (MDV) coding forglycoproteins B, C, D, and gp82 (gB, gC, gD, and UL32) and tegumentproteins UL47 and UL48, as well as genes from serotype 2 and 3 MDVcoding for glycoprotein B (gB2 and gB3). These recFPVs, alone and invarious combinations, including combinations of recFPV/MD-gBs withturkey herpesvirus (HVT) , were evaluated for ability to protectmaternal antibody positive (ab+) and negative (ab−) chickens againstchallenge with highly virulent MDV isolates. The protective efficacy wasalso compared to that of prototype MD vaccines. No protection wasinduced in ab+chickens by recFPV expressing gC, gD, UL47, or UL48. Incontrast, the recFPV/MD-gB construct protected about 23% of ab+ chickensagainst MDV challenge compared to 26% for cell-associated HVT. Levels ofprotection by recFPV/MD-gBs of different MDV serotypes was highest forgB, intermediate for gB2, and lowest for gB3. When recFPV/MD-gB wascombined with cell-associated HVT, protection was enhanced by an averageof 138% compared to the best component monovalent vaccine, and the meanlevel of protection was 59% compared to 67% for the HVT+SB-1 bivalentvaccine. Relatively high protection (50%) and enhancement (200%) wasalso observed between recFPV/MD-gB and cell-free HVT. These resultssuggest a specific synergistic interaction between recFPV/MD-gB and HVT,possibly analogous to that previously described between serotype 2 and 3viruses. Levels of protection by recFPV/MD-gB alone or by bivalentrecFPV/MD-gB+cell-associated HVT were similar to those of conventionalcell-associated MD vaccines. However, the bivalentrecFPV/MD-gB+cell-free HVT vaccine was clearly more protective thancell-free HVT alone and, thus, may be the most protective, entirelycell-free MD vaccine thusfar described.

[0154] Three serotypes of MDV have been described (2,6): serotype 1includes pathogenic isolates of chicken origin and their attenuatedderivatives, serotype 2 includes the naturally apathogenic isolates ofchicken origin, and serotype 3 includes the naturally apathogenicisolates of turkey origin known as turkey herpesvirus (HVT) (25).Vaccines derived from all three serotypes offer different levels ofprotection against the disease either alone or in bivalent and trivalentcombinations (24).

[0155] Glycoprotein B (gB) is highly conserved among herpesviruses. Inherpes simplex virus, it has been shown to be essential for virusinfectivity and thus involved in virus penetration in host cells (4,5).Antibodies to this glycoprotein are known to neutralize the virus (4,5).Recently, gBs from all three serotypes of MDV were shown to be alsohighly conserved (31) with predicted amino acid identities of 83 and 82%for gB2 and gB3 when compared with gB (12). Analysis of the gB complexof MDV with a neutralizing monoclonal antibody showed that it iscomposed of a precursor 100 kda molecular weight protein and twocleavage products of 60 kda and 49 kda glycoproteins (30). Thismonoclonal antibody recognized only the gB of serotype 1 and 3 and notthat of serotype 2 (15).

[0156] Preliminary trials indicated that recFPV/MD-gB induced highlevels of protection against MD challenge in maternal antibody negativechickens (9). Under the test conditions employed, the magnitude of theprotection induced by recFPV/MD-gB approached 100% but could not bedifferentiated from that induced by HVT.

[0157] We describe below the use of recFPVs expressing gB, otherglycoproteins, and tegument proteins of serotype 1 MDV as well as thoseexpressing gB from serotypes 2 and 3 of MDV, either alone or incombination with HVT, to protect against MD in chickens with MDVmaternal antibodies.

[0158] Viruses

[0159] Prototype vaccine viruses included the R2/23 (20) andCVI988/Rispens (10, 21) strains of serotype 1, the SB-1 (13) and 301B/1(17, 18) strains of serotype 2, and the FC126/2 (19) strain of HVT(serotype 3). Both cell-associated and cell-free stocks of HVT wereused. The cell-free HVT consisted of two different vaccine preparationsof the FC126 strain (Solvay Animal Health, Mendota Heights, MN). Allother vaccine stocks were prepared in the inventors' laboratory. Thevery virulent Md5 (26) and RB1B (14) strains of MDV were used aschallenge viruses. A large-plaque-forming clone (8) from acell-culture-propagated vaccine isolate of fowlpox virus (FPV) was usedas the parent virus to construct recombinant FPVs expressing MDV genes.

[0160] Construction of recombinant FPV

[0161] Cloning of MDV genes and construction of recFPVs were essentiallyas reported earlier (28). Recombinants were constructed that expressedgenes from serotype 1 Marek's disease virus (MDV) coding forglycoproteins B (gB) (12), C (gC) (3), D (gD) (11), gp82 (UL32), andtegument proteins UL47 and UL48 (29), as well as genes from serotype 2and 3 MDV coding for glycoprotein B (gB2 and gB3) (31). The sequenceTTTTTNT that has been reported to terminate early transcription inpoxvirus replication was modified in MDV genes whenever present by sitespecific mutagenesis (28). MDV genes were inserted into a multiplecloning site constructed within a nonessential region of FPV DNAdownstream from a strong synthetic pox virus promoter into pNZ1729Rinsertion vector (28). This vector contained the lacZ bacterial genewhich expresses the beta galactosidase of E. coli. The lacZ gene wasdownstream from a weak FPV promoter and in the opposite direction fromthe MDV gene.

[0162] Chicken embryo fibroblast (CEF) cultures infected for 5 hours ata multiplicity of infection of 5 with a vaccine isolate of FPV weretransfected with 10 μg of transfer vector containing different genes ofMDV. Using the expression of lacZ as an indicator, cloned recombinantviruses were identified and purified through several CEF passages. Purerecombinant virus was obtained after 4 serial passages of each clone.Two blind passages were made to ascertain the purity of eachrecombinant. Stocks of purified recFPV were stored at −70° C., titeredon CEF by plaque assay, and used for vaccination of chickens.

[0163] Chickens

[0164] Chickens were F₁ progeny of line 15I₅, males and 7₁ females. Formost experiments, these were from breeder hens vaccinated with all threeMD viral serotypes as described (18) and were considered positive formaternal antibodies (ab+). In one trial, chickens of the same F₁ crosswere from unvaccinated breeder hens that were free of antibodies to MDVand HVT; these were considered negative for maternal antibodies (ab−).All breeder chickens were maintained at the Avian Disease and OncologyLaboratory and were free of antibodies to avian leukosis virus,reticuloendotheliosis virus and various other poultry pathogens.

[0165] Protection trials

[0166] Groups of 12 to 17 chickens were vaccinated at 1 day of age with10⁶ plaque forming units (PFU) of recFPV vaccines by the wingweb (WW)route in trial 1 or by the intraabdominal (IA) route in all othertrials. Monovalent and polyvalent MDV and HVT vaccines were administeredto chickens by the IA route at a dose of 2000 PFU except where otherwiseindicated. The cell-free HVT used in trials 2 and 3 were from twodifferent batches. For polyvalent recFPV+MDV vaccines, each componentwas administered by a separate inoculation. For polyvalent vaccinescomposed of two MDV strains, the viruses were mixed and given as asingle inoculation. Ab+ chickens were used in trials 1-4; ab− chickenswere used in trial 5. Following vaccination, chickens were housed inmodified Horsfall-Bauer isolators.

[0167] At the 6th day post hatch, groups of vaccinated and unvaccinatedchickens were challenged by IA inoculation of 500 PFU of strain Md5(trials 1-4) or strain RB1B (trial 5). Mortality during the course ofthe experiment was recorded and chickens were examined for gross MDlesions. At about 56 days postchallenge (range 48-62), the remainingchickens were killed and examined for gross MD lesions.

[0168] The percent MD based on the number of chickens that died or werekilled and had gross MD lesions divided by the number of chickens atrisk (total less birds dying of other causes)×100 was determined foreach group. The percent protection was calculated for each vaccinatedgroup as the percent MD in unvaccinated, challenged controls less thepercent MD in the vaccinated, challenged group divided by the percent MDin the unvaccinated, challenged controls×100. The percent synergism wascalculated for each bivalent-vaccinated group as the percent protectionof the bivalent vaccine minus the percent protection of the better ofthe two constituent monovalent vaccines divided by the percentprotection of the best monovalent vaccine×100. Five trials wereconducted; each trial consisted of 2 or 3 replicates. Statisticalanalysis was performed on pooled data from all replicates. Differencesin percent protection were analyzed by computing interaction chi-squarevalues (16). The significance of percent synergism values was analyzedby comparing interaction chi-square values of the bivalent vaccine withthat of the best monovalent vaccine (22). Differences were considered tobe significant when p<0.05.

[0169] Comparative efficacy of recFPV expressing different MDV genes(Trial 1)

[0170] Chickens were immunized with five recFPVs with inserted serotype1 MDV genes encoding three different glycoproteins (gB, gC and gD) andtwo tegument proteins (UL47 and UL48). Mixtures of gB+gC andgB+gC+gD+UL47+UL48 were also tested. Other results, including thoseobtained with UL32, are shown in Tables 1 and 2. Conventional MDvaccines were included as controls. Results (Table 3) indicate thatrecFPV/MD-gB was the only recombinant among the five tested thatprovided significant protection. The use of other FPV recombinantsexpressing gC, gD, UL47 or UL48 genes in combination with therecFPV/MD-gB did not increase the percent protection. TABLE 3Vaccination trials to evaluate FPV recombinants expressing differentglycoproteins and tegument proteins of serotype 1 MDV (Trial 1)Replicate 1 Replicate 2 Replicate 3 Summary % % Pro- % % Pro- % % Pro- %% Pro- Vaccine^(A) N MD tection N MD tection N MD tection N MDtection^(B) recFPV monovalent: recFPV/MD-gB 15 73 27 17 88 12 17 82 1849 82 18 cd FPV/gC 16 100 0 17 100 0 17 100 0 50 100  0 a FPV/gD 17 1000 17 100 0 17 100 0 51 100  0 a FPV/UL47 14 93 7 16 100 0 17 100 0 47 98 2 ab FPV/UL48 14 93 7 15 100 0 15 93 7 44 96  5 abc recFPV polyvalent:recFPV/MD-gB + gC 15 67 33 17 82 18 16 100 0 48 83 17 cd recFPV/MD-gB +gC + gD + 17 94 6 17 71 29 17 94 6 51 86 13 bcd UL47 + UL48 Controls:HVT (CA) 17 41 59 17 88 12 17 100 0 51 77 24 d HVT (CA) + 301B/1 17 1882 17 63 35 17 29 71 51 32 63 e R2/23 17 24 76 17 41 59 11 27 73 45 5269 e None. 16 100 17 100 17 100 50 97

[0171] Protective synergism between recFPV/MD-gB and HVT (Trials 2 and3)

[0172] In trial 2, bivalent vaccines composed of recFPV/MD-gB withcell-associated HVT, cell-free HVT or CVI988/Rispens were evaluated forsynergism by comparison of the percent protection with that ofappropriate monovalent vaccines. Pooled data from three replicates(Table 4) show that percent protection was 8% for recFPV/MD-gB and 18%for cell-associated HVT but, when both were combined as a bivalentvaccine, protection was 66%, a 267% increase compared to cell-associatedHVT alone. Enhancement in individual replicates varied from 183 to 477%.Similarly, the recFPV/MD-gB combined with cell-free HVT protected 45% ofchickens compared to 8% for each of the individual components, a 462%increase (range 218 to >500). No synergism was apparent, however, whenrecFPV/MD-gB was combined with CVI988/Rispens.

[0173] Trial 3 included groups immunized with recFPV/MD-gB orrecFPV/gB3, both alone and in combination with cell-associated HVT. Theresults, presented in Table 5, resembled those of trial 2. Protection bythe recFPV/MD-gB+HVT vaccine (48%) was enhanced by 140% (range 72-173%in 3 replicates) compared to HVT alone (20%). The recFPV/gB3 constructfailed to provide any significant protection or synergism. TABLE 4Vaccination trials to evaluate synergism between the FPV/gB1 recombinantand MDV vaccines of serotypes 1 and 3 (Trial 2). Replicate 1 Replicate 2Replicate 3 Summary % % Pro- % % Pro- % % Pro- % % Pro- % Vaccine^(A) NMD tection N MD tection N MD tection N MD tection^(B) Synergism^(C)recFPV monovalent: recFPV/MD-gB 17 88 12 17 100 0 17 88 12 51 92  8 arecFPV Polyvalent: recFPV/MD-gB + FC126 (CA) 17 49 51 16 25 75 17 35 6550 43 66 cd 267* recFPV/MD-gB + FC126 (CF) 16 50 51 16 75 25 17 41 59 4955 45 b 462* recFPV/MD-gB + CVI988/ 17 11 88 17 11 88 17 43 56 50 22 78cde  <1 ns Rispens Controls: HVT (CA) 17 82 18 16 87 13 17 76 24 50 8218 b HVT (CF) 17 88 16 16 100 0 17 88 12 50 92  8 a HVT (CA) + SB-1 16 694 17 18 22 17 29 71 50 18 82 de HVT (CF) + SB-1 17 17 83 16 43 56 17 4753 50 36 64 bcd HVT (CA) + 301B/1 17 11 88 13 23 77 17 0 100 47 11 89 eCVI988/Rispens 16 6 94 17 12 88 17 51 88 47 38 90 e R2/23 13 0 100 17 1253 17 12 47 50 10 62 bc None 17 100 — 17 100 — 17 100 — 51 100 —

[0174] TABLE 5 Vaccination trials to evaluate synergism betweendifferent FPV/gB recombinants and HVT, and the comparative efficacy ofdifferent FPV/gB recombinants (Trial 3). Replicate 1 Replicate 2Replicate 3 Summary % % Pro- % % Pro- % % Pro- % % Pro- % Vaccine^(A) NMD tection N MD tection N MD tection N MD tection^(B) Synergism^(C)recFPV Monovalent: recFPV/MD-gB 15 73 27 17 82 18 17 88 12 49 82 18 brecFPV/MD-gB2 17 53 47 17 47 53 17 94 6 51 65 35 bc recFPV/MD-gB3 15 1000 16 100 0 17 94 6 48 98  2 a recFPV Polyvalent: recFPV/MD-gB + HVT (CA)15 47 53 14 50 50 17 59 41 46 52 48 cd 140* recFPV/MD-gB3 + HVT (CA) 1464 36 17 88 12 17 59 41 48 71 29 bc  45 ns Controls: HVT (CA) 5 80 20 1771 29 34 85 15 56 80 20 b HVT (CA) + SB-1 17 29 71 17 35 65 17 53 47 5139 61 de CVI988/Rispens 17 41 59 17 24 76 17 41 59 51 35 65 de None 14100 17 100 17 100 48 100

[0175] Efficacy of conventional vaccines

[0176] Conventional cell-associated vaccines were used as controls intrials 1-3. Protection was least with cell-free HVT, intermediate withcell-associated HVT, and greatest with bivalent serotype 2+3 or serotype1 vaccines. The relative efficacy of these vaccines was consistent withprevious observations.

[0177] Dosage and administration of the vaccine

[0178] The vaccine described above can be administered in a variety ofdifferent ways:

[0179] 1. By inoculation of recFPV/gB at a dose of 10² to 10⁶ PFU perchick, more preferably at a dose of 1 to 10⁶ PFU per chick, given byeither the intraabdominal, wingweb, intramuscular, or subcutaneous routeplus separate inoculation of cell-associated HVT vaccine at a dose of500 to 20,000 PFU, more preferably at a dose of 2,000 to 10,000 PFU, bythe intraabdominal, intramuscular, or subcutaneous route;

[0180] 2. As in 1, above, except that cell-free HVT vaccine issubstituted for cell-associated HVT vaccine;

[0181] 3. As in 1, above, except that the recFPV/gB and HVT are combinedin the same inoculum;

[0182] 4. As in 1, above, except that the recFPV/gB may be repalced byrecFPV vaccines expressing gB plus other inserted genes from MDV orother sources; or

[0183] 5. As in 1, above, when the HVT is derived from any of therecognized strains, including FC126, that can be classified as aserotype 3 Marek's disease virus.

[0184] The invention being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the present invention, andall such modifictions as would be obvious to one skilled in the art areintended to be included within the scope of the following claims.

References Cited In Example 9

[0185] 1. Brunovskis et al. In: 4th International Symposium on Marek'sDisease, 19th World's Poultry Congress Vol. 1, World's Poultry ScienceAssn., Amsterdam, pp. 118-122 (1992).

[0186] 2. Bulow et al. Avian Pathology, 4:133-146 (1975).

[0187] 3. Coussens et al. J. Virol. 62:467-476 (1988).

[0188] 4. Glorioso et al. J. Virol. 50:805-812 (1984).

[0189] 5. Highlander et al. J. Virol. 63:730-738 (1989).

[0190] 6. Lee et al. J. Immunol. 130:1003-1006 (1983).

[0191] 7. Nazerian, K. In: Viral Oncolocy, G. Klein, Ed., pp. 665-682(1980).

[0192] 8. Nazerian et al. Avian Dis. 33:458-465 (1989).

[0193] 9. Nazerian et al. J. Virol. 66:1409-1413 (1992).

[0194] 10. Rispens et al. Avian Dis. 16:108-125 (1972).

[0195] 11. Ross et al. J. Gen. Virol. 72: 949-954 (1991).

[0196] 12. Ross et al. J. Gen. Virol. 70:1789-1804 (1989).

[0197] 13. Schat et al. J. Nat'l. Cancer Inst. 60:1075-1082 (1978).

[0198] 14. Schat et al. Avian Pathology 11:593-605 (1982).

[0199] 15. Silva et al. Virology 136:307-320 (1984).

[0200] 16. Steal et al. Principles and Procedures of Statistics,McGraw-Hill Book Company, Inc., New York, (1960).

[0201] 17. Witter Avian Dis. 27:113-132 (1983).

[0202] 18. Witter Avian Dis., 31:752-765 (1987).

[0203] 19. Witter In: Advances In Marek's Disease Research, S. Kato etal., Eds., Japanese Association on Marek's Disease, Osaka, Japan, pp.398-404 (1988).

[0204] 20. Witter Avian Dis. 35:877-891 (1991).

[0205] 21. Witter In: 4th International Symposium on Marek's Disease,19th World's Poultry Congress. Vol. 1., World's Poultry Science Assoc.,Amsterdam, pp. 315-319 (1992).

[0206] 22. Witter Avian Pathology 21:601-614 (1992).

[0207] 23. Witter Avian Pathology 8:145-156 (1979).

[0208] 24. Witter et al. Avian Pathology 13:75-92 (1984).

[0209] 25. Witter et al. Am J. Vet. Res. 31:525-538 (1970).

[0210] 26. Witter et al. Avian Dis. 24:210-232 (1980).

[0211] 27. Yanagida et al. In: 4th International Symposium on Marek'sDisease, 19th World's Poultry Congress. Vol. 1., World's Poultry ScienceAssn., Amsterdam, pp. 44-48 (1992).

[0212] 28. Yanagida et al. J. Virol. 66:1402-1408 (1992).

[0213] 29. Yanagida et al. J. Gen. Virol. 74:1837-1845 (1993).

[0214] 30. Yoshida et al. Gene 150:303-306 (1994).

[0215] 31. Yoshida et al. Virology 200:484-493 (1994).

EXAMPLE 10 Construction of Transfer vector PNZ29RMDgEcI

[0216] The MDV gE gene was cloned from genomic DNA of MDV strain GA byPCR. The oligonucleotide primers used for the PCR are5′-GGGGAGATCTCATAATGTGTGTTTTCCAAATC-3′ (pgE-1, SEQ ID NO:24, BglII siteunderlined) and 5′-GGGGGTCGACGTCCATATACTATATCCC-3′ (pgE-2, SEQ ID NO:25,SalI site underlined). Primers pgE-1 and pgE-2 comprise the nucleotidesequence of the 5′ terminus and 3′ flanking region of the MDV gE gene,respectively (Brunnovskis et al., Virology 206:324-338 (1995)). A 1,558bp DNA fragment amplified by the PCR was digested with BglII and SalI.The BglII-SaII fragment was cloned into BamHI/SalI digested pGTPs. Theresulting plasmid was named pGTPsgE.

[0217] The MDV gI gene was cloned from genomic DNA of MDV strain GA byPCR. The oligonucleotide primers used for the PCR are5′-GGGGAGATCTGCGATGTATGTACTACAATTA-3′ (pgI-1, SEQ ID NO:26, BglII siteunderlined) and 5′-CTAACAGGTACCACCTACCTATAA-3′ (pgI-2, SEQ ID NO:27,KpnI site underlined). Primers pgI-1 and pgI-2 comprise the nucleotidesequence of the 5′ terminus and 3′ flanking region of the MDV gE gene,respectively (Brunnovskis et al., Virology 206:324-338 (1995)). A 1,118bp DNA fragment amplified by the PCR was digested with KpnI, and thenpartially digested with BglII. The BglII-KpnI fragment was cloned intoBamHI/KpnI digested pGTPs. The resulting plasmid was named pGTPsgI. A1,669 bp BglI fragment from pGTPs-gE was cloned into SfiI digestedpNZ1829R. The resulting plasmid was named pNZ29RMDgE. A 1,232 bp BglIfragment from pGTPsgI was cloned into SfiI digested pNZ29RMDgE to obtaintransfer vector pNZ29RMDgEgI.

EXAMPLE 11 Construction of Transfer vectors PNZ29RMDqBqEqI andPNZ29RMDqBqEqIUL32

[0218] A 1,669 bp BglI fragment from pGTPsgE was cloned into SfiIdigested pNZ29RMDgBSfi. The resulting plasmid was named pNZ29RMDgBgE. A1,232 bp BglI fragment from pGTPsgI was cloned into SfiI digestedpNZ29RMDgE to obtain transfer vector pNZ29RMDgBgEgI. A 2,061 bp BglIfragment from pGTPsgI was cloned into SfiI digested pNZ29RMDgBgEgI toobtain transfer vector pNZ29RMDgBgEgIUL32.

EXAMPLE 12 Generation of Recombinant FPVs recFPV/MD-gE/gI,recFPV/MD-gB/gE/gI and recFPV/MD-gB/gE/gI/UL32

[0219] Recombinants recFPV/MD-gE/gI, recFPV/MD-gB/gE/gI andrecFPV/MD-gB/gE/gI/UL32 were generated by electroporation of thetransfer vectors pNZ29RMDgEgI, pNZ29RMDgBgEgI and pNZ29RMDgBgEgIUL32,respectively, into host cells according to the methods described inEXAMPLE 5.

EXAMPLE 13 Protection of Antibody-Negative Chickens with RecombinantFPVs Expressing the gB Gene, or the gE and gI Genes aQainst a veryvirulent Strain of MDV (Md5)

[0220] Three sets of experiments were performed. Separate groups of1-day-old chickens from 15×7 AB (−) chickens line susceptible to Marek'sDisease (MD) were vaccinated with 10⁵ PFU of recFPV/MD-gB (U.S. Pat. No.5,369,025; Example 4), 10⁵ PFU of recFPV/MD-gE/gI (Example 12), 10⁵ PFUof recFPV/MD-gBgE/gI (Example 12) or 10⁵ PFU of the USDA strain of FPV(Nazerian et al., Avian Disease 33, 458-465, (1989)). Another group ofsimilar chickens was kept unvaccinated. All were kept in strictisolation. At 6 days of age all chickens were challenged with 5×10² PFUof the very virulent Md5 strain of MDV. A sixth group of chickens wasneither vaccinated nor challenged. Mortality caused by MD was recordedduring the trial, and the end of the 8 week trial, all chickens wereexamined for gross lesions and tumors typical of MD. The results of thisstudy are presented in Table 6.

[0221] One hundred percent of unvaccinated chickens and 98% of thechickens vaccinated with the USDA of FPV died of MD before the end ofthe trial. On the other hand, chickens vaccinated with recFPV/MD-gE/gIwere significantly well protected (PI=58%), which was similar protectionobserved (PI=63%) of those vaccinated with recFPV/MD-gB. Furthermore,recFPV expressing the gB, gE and gI genes conferred better protection(PI=75%). TABLE 6 Vaccination trials to evaluate FPV recombinantsexpressing the gB, gE and gI genes of MDV in Ab(−) chickens challengedwith the Md5 isolate of MDV at 5 days post vaccination Trial 1 Trial 2Trial 3 Summary Vaccines MD test % MD PI MD test % MD PI MD test % MD PIMD test % MD PI recFPV/MD-gB 7 17 41 59 3 17 18 82 9 17 53 47 19 51 3763 recFPV/MD-gE/gI 6 16 38 62 8 17 47 53 7 17 41 59 21 50 42 58recFPV/MD-gB/gE/gI 2 17 12 88 6 17 35 65 5 17 29 71 13 51 25 75 FPV 1616 100 0 13 14 93 7 16 16 100 0 45 46 98 2 None 16 16 100 15 15 100 1515 100 46 46 100 None (no chal.) 0 5 0 0 5 0 0 5 0 0 15 0

EXAMPLE 14 Protection of Antibody-Positiive Chickens with RecombinantFPVs Expressing the gB, gE and gI Genes, or the gB, gE, gI and UL32Genes Against a Very Virulent Strain of MDV (Md5)

[0222] Three sets of experiments were performed. Separate groups of1-day-old chickens from the 15×7 Ab (+) chicken line susceptible to MDwere vaccinated with 10⁵ PFU of recFPV/MD-gB (U.S. Pat. No. 5,369,025;Example 4), 10⁵ PFU of recFPV/MD-gBgE/gI (Example 12), 10⁵ PFU ofrecFPV/MD-gBgE/gI/UL32 (Example 12) or 2×10³ PFU of the FC126 strain ofHVT (cell-associated). Another group,of similar chickens was keptunvaccinated. All were kept in strict isolation. At 6 days of age allchickens were is challenged with 5×10² PFU of the very virulent MdSstrain of MDV. Mortality caused by MD was recorded during the trial, andat the end of the 8 week trial, all chickens were examined for grosslesions and tumors typical of MD. The results of this study arepresented in Table 7.

[0223] One hundred percent of unvaccinated chickens died of MD beforethe end of the trial. Forty-three percent of chickens vaccinated withrecFPV/MD-gB were protected from lesions, compared to 44% of thosevaccinated with HVT. The level of protection by recFPV/MD-gB/gE/gI/UL32(PI=66%) was slightly higher than that of recFPV/MD-gB/gE/gI (PI=53%).TABLE 7 Vaccination trials to evaluate FPV recombinants expressing thegB, gB, gI and UL32 genes of MDV in Ab(+)chickens challenged with theMd5 isolate of MDV at 5 days post vaccination Trial 1 Trial 2 Trial 3Summary Vaccines MD test % MD PI MD test % MD PI MD test % MD PI MD test% MD PI recFPV/MD-gB 11 17 65 35 8 17 47 53 10 17 59 41 29 51 57 43recFPV/MD-gB/gB/gI 6 16 38 62 9 16 56 44 8 17 47 53 23 49 47 53recFPV/MD-gB/gB/gI/ 5 17 29 71 5 17 29 71 7 16 44 56 17 50 34 66 UL32HVT 9 17 53 47 9 17 53 47 10 16 63 37 28 50 56 44 None 16 16 100 17 17100 16 16 100 49 49 100

[0224]

1 29 1925 base pairs nucleic acid single linear DNA (genomic) NO NO 1ATGGCCAACC GCCCTACAGA GTTGGCAGCT TTTATCCGAT CTTCTGGAGA AGCAGATGGA 60TGGATAGAGG AGTCCTTCAA AGAACCCTAT GTGGCATTTA ATCCGGACGT CTTGATGTA 120AATGACACGC TTTTTAACGA GTTATTACTC TCCGCCCACG CGCTCAAGAT CAACAGTAT 180CAGGATGTTC AGAGTGATGA TACCGTGGAG GATGCGGGAG ATATTGGGAA TGAAGTTAT 240CATTCGGAAT TAGTAACTTT TATAGAGACT GCTGCAGATG TTTATGCCTT AGATCGTCA 300TGCCTTGTTT GTCGTGTGCT AGATATGTAC AGGCGCAATT TCGGTTTATC AGCTCTATG 360ATGGCAGATT ATGCTTTTTT ATGTTCCAAA TGTCTTGGTT CTCCACCATG TGCAACTGC 420ACCTTTATAG CCGCGTTTGA ATTCGTATAT ATAATGGATA AACACTTTCT ATCCGATCA 480GGTTGTACAC TCGTACGCTC CTTTGGAAAA AAACTTTTAA CTCTCGAAGA TATTCAGAG 540CATTTTTTTC TGCATGGCTG TTTTCGAACG GACGGGGGCG TTCCTGGACG ACGCCATGA 600GAAGTTATTA CGTCTCGTTC TAAGCAAGGA CGATTAGTAG GGCGACGTGG GAAATTTTC 660ACTGCGGGTG ATGCCAAAGT CTTGTACAGT AATTACTCAT ATTTAGCTCA GAGTGCTAC 720CGAGCCCTGT TAATGACCTT ATCTGATTTA GGTTCTGCAC CGCTAGAAGT TATCGAAGG 780CGACAAAAGT CTATTTCGGG GGATGTTCGA AATGAGTTGA GGGATGGCAT AGAGAGTAG 840AAAAGGGTCG CGCATGTCAT TCATTCCGTT GGACCAGTCC ACTCATGCCC AACTACTCT 900TCCGTTGCTT TGGCGGGCTG GAAAGATTGT GCTAAAAACG TAGAATGTAA CTTTTTTCA 960CTGGAAAGTT GTACTTTGCG CGCATCGTCC GAGGATAATG ATTATGAACA CGAGTGGG 1020CTCCGAGCAA GTGAAGAAAA GTTAAATGTG GTGGAAAATG TTCAGGACAT GCAACAGA 1080GATGCGTCTC AATGCGAACA TCATGAACAT GCAAGAAATG AGGATTGTAC AATGGGTT 1140GGCAACCTCG TTTTATTGTT ATTAGCGGGA ACGGGGTCTG CACCTGAGGC AGCGAGCG 1200CTCGCATTCA TGGCCGCAAA AGTTAGAAGG GAAACGGTGG ATATATTTTG GAAAAATC 1260AGAAGGGAAT TTGCTAATGA CGTTACTGCA GCATACAGTG CATGTTACGG TGAGGATT 1320GAACCCGATT TAGAGTTAGG CCCATTGATG ATAACACAGT TAAAGCACGC GATAACAA 1380GGAGGAACAT CTGCGGAGTG TTTATTATGT AACCTGCTGC TGATACGTAC ATATTGGC 1440GCAATGCGTA AATTTAAACG CGATATCATC ACATATTCCG CCAACAATAT AGGTTTAT 1500CATAGCATAG AACCTGTTCT AGATGCTTGG CGATCACAGG GACATCGTAC AGATTTGG 1560GACGAAGGAT GTTTTGTAAC ATTAATGAAA AGCGCGGGAA CGGAGGCCAT TTATAAAC 1620CTATTCTGCG ATCCAATGTG TGCGGCACGA ATAGCCCAGA CCAATCCACG ATCGTTAT 1680GATCACCCAG ATGCCACCAA TCATGACGAA CTAGCATTAT ATAAAGCCCG TCTCGCCA 1740CAGAACCATT TTGAAGGTCG CGTATGTGCT GGACTTTGGG CTTTGGCGTA TACGTTTA 1800ACTTATCAGG TCTTTCCTCC CCGTCAACCG CACTGTCTGC TTTCGTTAAA GACGCTGG 1860CATTGTTGCA AAGACATTCC ATCTCCTTGA TATCTCTCGA GCATACATTA GGAGTCTA 1920TGTAA 1925 641 amino acids amino acid single linear DNA (genomic) NO NO2 Met Ala Asn Arg Pro Thr Glu Leu Ala Ala Phe Ile Arg Ser Ser Gl 1 5 1015 Glu Ala Asp Gly Trp Ile Glu Glu Ser Phe Lys Glu Pro Tyr Val Al 20 2530 Phe Asn Pro Asp Val Leu Met Tyr Asn Asp Thr Leu Phe Asn Glu Le 35 4045 Leu Leu Ser Ala His Ala Leu Lys Ile Asn Ser Ile Gln Asp Val Gl 50 5560 Ser Asp Asp Thr Val Glu Asp Ala Gly Asp Ile Gly Asn Glu Val Il 65 7075 80 His Ser Glu Leu Val Thr Phe Ile Glu Thr Ala Ala Asp Val Tyr Al 8590 95 Leu Asp Arg Gln Cys Leu Val Cys Arg Val Leu Asp Met Tyr Arg Ar 100105 110 Asn Phe Gly Leu Ser Ala Leu Trp Met Ala Asp Tyr Ala Phe Leu Cy115 120 125 Ser Lys Cys Leu Gly Ser Pro Pro Cys Ala Thr Ala Thr Phe IleAl 130 135 140 Ala Phe Glu Phe Val Tyr Ile Met Asp Lys His Phe Leu SerAsp Hi 145 150 155 160 Gly Cys Thr Leu Val Arg Ser Phe Gly Lys Lys LeuLeu Thr Leu Gl 165 170 175 Asp Ile Gln Arg His Phe Phe Leu His Gly CysPhe Arg Thr Asp Gl 180 185 190 Gly Val Pro Gly Arg Arg His Asp Glu ValIle Thr Ser Arg Ser Ly 195 200 205 Gln Gly Arg Leu Val Gly Arg Arg GlyLys Phe Ser Thr Ala Gly As 210 215 220 Ala Lys Val Leu Tyr Ser Asn TyrSer Tyr Leu Ala Gln Ser Ala Th 225 230 235 240 Arg Ala Leu Leu Met ThrLeu Ser Asp Leu Gly Ser Ala Pro Leu Gl 245 250 255 Val Ile Glu Gly ArgGln Lys Ser Ile Ser Gly Asp Val Arg Asn Gl 260 265 270 Leu Arg Asp GlyIle Glu Ser Arg Lys Arg Val Ala His Val Ile Hi 275 280 285 Ser Val GlyPro Val His Ser Cys Pro Thr Thr Leu Ser Val Ala Le 290 295 300 Ala GlyTrp Lys Asp Cys Ala Lys Asn Val Glu Cys Asn Phe Phe Gl 305 310 315 320Leu Glu Ser Cys Thr Leu Arg Ala Ser Ser Glu Asp Asn Asp Tyr Gl 325 330335 His Glu Trp Glu Leu Arg Ala Ser Glu Glu Lys Leu Asn Val Val Gl 340345 350 Asn Val Gln Asp Met Gln Gln Ile Asp Ala Ser Gln Cys Glu His Hi355 360 365 Glu His Ala Arg Asn Glu Asp Cys Thr Met Gly Tyr Gly Asn LeuVa 370 375 380 Leu Leu Leu Leu Ala Gly Thr Gly Ser Ala Pro Glu Ala AlaSer Gl 385 390 395 400 Leu Ala Phe Met Ala Ala Lys Val Arg Arg Glu ThrVal Asp Ile Ph 405 410 415 Trp Lys Asn His Arg Arg Glu Phe Ala Asn AspVal Thr Ala Ala Ty 420 425 430 Ser Ala Cys Tyr Gly Glu Asp Ser Glu ProAsp Leu Glu Leu Gly Pr 435 440 445 Leu Met Ile Thr Gln Leu Lys His AlaIle Thr Lys Gly Gly Thr Se 450 455 460 Ala Glu Cys Leu Leu Cys Asn LeuLeu Leu Ile Arg Thr Tyr Trp Le 465 470 475 480 Ala Met Arg Lys Phe LysArg Asp Ile Ile Thr Tyr Ser Ala Asn As 485 490 495 Ile Gly Leu Phe HisSer Ile Glu Pro Val Leu Asp Ala Trp Arg Se 500 505 510 Gln Gly His ArgThr Asp Leu Gly Asp Glu Gly Cys Phe Val Thr Le 515 520 525 Met Lys SerAla Gly Thr Glu Ala Ile Tyr Lys His Leu Phe Cys As 530 535 540 Pro MetCys Ala Ala Arg Ile Ala Gln Thr Asn Pro Arg Ser Leu Ph 545 550 555 560Asp His Pro Asp Ala Thr Asn His Asp Glu Leu Ala Leu Tyr Lys Al 565 570575 Arg Leu Ala Ser Gln Asn His Phe Glu Gly Arg Val Cys Ala Gly Le 580585 590 Trp Ala Leu Ala Tyr Thr Phe Lys Thr Tyr Gln Val Phe Pro Pro Ar595 600 605 Xaa Thr Ala Leu Ser Ala Phe Val Lys Asp Ala Gly Ala Leu LeuGl 610 615 620 Arg His Ser Ile Ser Leu Ile Ser Leu Glu His Thr Leu GlyVal Ty 625 630 635 640 Val 91 base pairs nucleic acid single linear DNA(genomic) NO NO 3 TTTTTTTTTT TTTTTTTTTT GGCATATAAA TAATAAATAC AATAATTAATTACGCGTAAA 60 AATTGAAAAA CTATTCTAAT TTATTGCACT C 91 9 base pairs nucleicacid single linear DNA (genomic) NO NO 4 GGYCAATCT 9 23 base pairsnucleic acid single linear DNA (genomic) NO NO 5 AGCTGCCCCC CCGGCAAGTTGCA 23 17 base pairs nucleic acid single linear DNA (genomic) NO NO 6TCGACATTTT TATGTAC 17 22 base pairs nucleic acid single linear DNA(genomic) NO NO 7 AATTCGGCCG GGGGGGCCAG CT 22 14 base pairs nucleic acidsingle linear DNA (genomic) NO NO 8 GGCCCCCCCG GCCG 14 26 base pairsnucleic acid single linear DNA (genomic) NO NO 9 CCCCGGATCC GGCCATGGCCAACCGC 26 24 base pairs nucleic acid single linear DNA (genomic) NO YES10 AAGAATGCAT AATCTGCCAT CCAT 24 26 base pairs nucleic acid singlelinear DNA (genomic) NO NO 11 GATTATGCAT TCTTATGTTC CAAATC 26 33 basepairs nucleic acid single linear DNA (genomic) NO YES 12 ACAGCCATGGAGAAAGAAAT GTCTCTGAAT ATC 33 23 base pairs nucleic acid single linearDNA (genomic) NO NO 13 TTCTCCATGG CTGTTTTCGA ACG 23 30 base pairsnucleic acid single linear DNA (genomic) NO YES 14 CCCCGTCGAC TTACACGTAGACTCCTAATG 30 15 base pairs nucleic acid single linear DNA (genomic) NONO 15 TATGCTTTTT TATGT 15 15 base pairs nucleic acid single linear DNA(genomic) NO NO 16 TATGCATTCT TATGT 15 5 amino acids amino acid singlelinear peptide NO NO 17 Tyr Ala Phe Leu Cys 1 5 15 base pairs nucleicacid single linear DNA (genomic) NO NO 18 TATGCTTTTT TATGT 15 18 basepairs nucleic acid single linear DNA (genomic) NO NO 19 CATTTTTTTCTGCATGGC 18 18 base pairs nucleic acid single linear DNA (genomic) NO NO20 CATTTCTTTC TCCATGGC 18 6 amino acids amino acid single linear peptideNO NO 21 His Phe Phe Leu His Gly 1 5 14 base pairs nucleic acid singlelinear DNA (genomic) NO NO 22 GGCCCCCCCG GCCG 14 22 base pairs nucleicacid single linear DNA (genomic) NO NO 23 AATTCGGCCGG GGGGGCCAGC T 22 32base pairs nucleic acid single not relevant other nucleic acid /desc =“pgE-1 PCR primer for MDV gE NO NO 24 GGGGAGATCT CATAATGTGT GTTTTCCAAATC 32 28 base pairs nucleic acid single not relevant other nucleic acid/desc = ”pgE-2 PCR primer for MDV gE NO YES 25 GGGGGTCGAC GTCCATATACTATATCCC 28 31 base pairs nucleic acid not relevant not relevant othernucleic acid /desc = “pgGI-1 PCR primer for MDV NO NO 26 GGGGAGATCTGCGATGTATG TACTACAATT A 31 24 base pairs nucleic acid single notrelevant other nucleic acid /desc = ”pgI-2 PCR primer for MDV gI NO YES27 CTAACAGGTA CCACCTACCT ATAA 24 355 amino acids amino acid not relevantlinear protein NO internal Marek′s disease virus type I GA Protein1..355 /label= protein /note= “gI protein” 28 Met Tyr Val Leu Gln LeuLeu Phe Trp Ile Arg Leu Phe Arg Gly Il 1 5 10 15 Trp Ser Ile Val Tyr ThrGly Thr Ser Val Thr Leu Ser Thr Asp Gl 20 25 30 Ser Ala Leu Val Ala PheCys Gly Leu Asp Lys Met Val Asn Val Ar 35 40 45 Gly Gln Leu Leu Phe LeuGly Asp Gln Thr Arg Thr Ser Ser Tyr Th 50 55 60 Gly Thr Thr Glu Ile LeuLys Trp Asp Glu Glu Tyr Lys Cys Tyr Se 65 70 75 80 Val Leu His Ala ThrSer Tyr Met Asp Cys Pro Ala Ile Asp Ala Th 85 90 95 Val Phe Arg Gly CysArg Asp Ala Val Val Tyr Ala Gln Pro His As 100 105 110 Arg Val Gln ProPhe Pro Glu Lys Gly Thr Leu Leu Arg Ile Val Gl 115 120 125 Pro Arg ValSer Asp Thr Gly Ser Tyr Tyr Ile Arg Val Ala Leu Al 130 135 140 Gly ArgAsn Met Ser Asp Ile Phe Arg Met Ala Val Ile Ile Arg Se 145 150 155 160Ser Lys Ser Trp Ala Cys Asn His Ser Ala Ser Ser Phe Gln Ala Hi 165 170175 Lys Cys Ile Arg Tyr Val Asp Arg Met Ala Phe Glu Asn Tyr Leu Il 180185 190 Gly His Val Gly Asn Leu Leu Asp Ser Asp Ser Glu Leu His Ala Il195 200 205 Tyr Asn Ile Thr Pro Gln Ser Ile Ser Thr Asp Ile Asn Ile IleTh 210 215 220 Thr Pro Phe Tyr Asp Asn Ser Gly Thr Ile Tyr Ser Pro ThrVal Ph 225 230 235 240 Asn Leu Phe Asn Asn Asn Ser His Val Asp Ala MetAsn Ser Thr Gl 245 250 255 Met Trp Asn Thr Val Leu Lys Tyr Thr Leu ProArg Leu Ile Tyr Ph 260 265 270 Ser Thr Met Ile Val Leu Cys Ile Ile AlaLeu Ala Ile Tyr Leu Va 275 280 285 Cys Glu Arg Cys Arg Ser Pro His ArgArg Ile Tyr Ile Gly Glu Pr 290 295 300 Arg Ser Asp Glu Ala Pro Leu IleThr Ser Ala Val Asn Glu Ser Ph 305 310 315 320 Gln Tyr Asp Tyr Asn ValLys Glu Thr Pro Ser Asp Val Ile Glu Ly 325 330 335 Glu Leu Met Glu LysLeu Lys Lys Lys Val Glu Leu Leu Glu Arg Gl 340 345 350 Glu Cys Val 355497 amino acids amino acid not relevant linear protein NO internalMarek′s disease virus type I GA Protein 1..497 /label= protein /note=“gE protein” 29 Met Cys Val Phe Gln Ile Leu Ile Ile Val Thr Thr Ile LysVal Al 1 5 10 15 Gly Thr Ala Asn Ile Asn His Ile Asp Val Pro Ala Gly HisSer Al 20 25 30 Thr Thr Thr Ile Pro Arg Tyr Pro Pro Val Val Asp Gly ThrLeu Ty 35 40 45 Thr Glu Thr Trp Thr Trp Ile Pro Asn His Cys Asn Glu ThrAla Th 50 55 60 Gly Tyr Val Cys Leu Glu Ser Ala His Cys Phe Thr Asp LeuIle Le 65 70 75 80 Gly Val Ser Cys Met Arg Tyr Ala Asp Glu Ile Val LeuArg Thr As 85 90 95 Lys Phe Ile Val Asp Ala Gly Ser Ile Lys Gln Ile GluSer Leu Se 100 105 110 Leu Asn Gly Val Pro Asn Ile Phe Leu Ser Thr LysAla Ser Asn Ly 115 120 125 Leu Glu Ile Leu Asn Ala Ser Leu Gln Asn AlaGly Ile Tyr Ile Ar 130 135 140 Tyr Ser Arg Asn Gly Asp Glu Asp Cys LysLeu Asp Val Val Val Va 145 150 155 160 Gly Val Leu Gly Gln Ala Arg AspArg Leu Arg Gln Met Ser Ser Pr 165 170 175 Met Ile Ser Ser His Ala AspIle Lys Leu Ser Leu Lys Asn Phe Ly 180 185 190 Ala Leu Val Tyr His ValGly Asp Thr Ile Asn Val Ser Thr Ala Va 195 200 205 Ile Leu Gly Pro SerPro Glu Ile Phe Thr Leu Glu Phe Arg Val Le 210 215 220 Phe Leu Arg TyrAsn Pro Thr Cys Lys Phe Val Thr Ile Tyr Glu Pr 225 230 235 240 Gly IlePhe His Pro Lys Glu Pro Glu Gly Ile Thr Thr Ala Glu Gl 245 250 255 SerVal Cys His Phe Ala Ser Asn Ile Asp Ile Leu Gln Ile Ala Al 260 265 270Ala Arg Ser Glu Asn Cys Ser Thr Gly Tyr Arg Arg Cys Ile Tyr As 275 280285 Thr Ala Ile Asp Glu Ser Val Gln Ala Arg Leu Thr Phe Ile Glu Pr 290295 300 Gly Ile Pro Ser Phe Lys Met Lys Asp Val Gln Val Asp Asp Ala Gl305 310 315 320 Leu Tyr Val Val Val Ala Leu Tyr Asn Gly Arg Pro Ser AlaTrp Th 325 330 335 Tyr Ile Tyr Leu Ser Thr Val Glu Thr Tyr Leu Asn ValTyr Glu As 340 345 350 Tyr His Lys Pro Gly Phe Gly Tyr Lys Ser Phe LeuGln Asn Ser Se 355 360 365 Ile Ile Asp Glu Asp Glu Ala Ser Asp Trp SerSer Ser Ser Ile Ly 370 375 380 Arg Arg Asn Asn Gly Thr Ile Leu Tyr AspIle Leu Leu Thr Ser Le 385 390 395 400 Ser Ile Gly Ala Ile Ile Ile ValIle Val Gly Gly Val Cys Ile Al 405 410 415 Ile Leu Ile Arg Arg Arg ArgArg Arg Arg Thr Arg Gly Leu Phe As 420 425 430 Glu Tyr Pro Lys Tyr MetThr Leu Pro Gly Asn Asp Leu Gly Gly Me 435 440 445 Asn Val Pro Tyr AspAsn Ala Cys Ser Gly Asn Gln Val Glu Tyr Ty 450 455 460 Gln Glu Lys SerAsp Lys Met Lys Arg Met Gly Ser Gly Tyr Thr Al 465 470 475 480 Trp LeuLys Asn Asp Met Pro Lys Ile Arg Lys Arg Leu Asp Leu Ty 485 490 495 His

1. An isolated, purified DNA molecule comprising the nucleotide sequenceshown in SEQ ID NO:1, or a nucleotide sequence biologically functionallyequivalent thereto.
 2. An isolated, purified DNA molecule comprising anucleotide sequence encoding a polypeptide having the amino acidsequence shown in SEQ ID NO:2, or encoding a polypeptide biologicallyfunctionally equivalent thereto.
 3. An isolated, purified polypeptidehaving the amino acid sequence shown in SEQ ID NO:2, or a polypeptidebiologically functionally equivalent thereto.
 4. A recombinant vectorcomprising said DNA molecule of claim
 2. 5. The recombinant vector ofclaim 4, wherein said recombinant vector is a virus vector.
 6. Arecombinant virus that expresses said DNA molecule of claim
 2. 7. Therecombinant virus of claim 6, selected from the group consisting of arecombinant poxvirus, a recombinant avian herpesvirus, a recombinantadenovirus, and a recombinant influenza virus.
 8. The recombinant virusof claim 7, selected from the group consisting of a recombinant raccoonpoxvirus, a recombinant vaccinia virus, a recombinant pigeon poxvirus, arecombinant fowlpox virus, a recombinant Marek's disease virus, arecombinant canary poxvirus, a recombinant quail poxvirus, a recombinantturkey poxvirus, a recombinant turkey herpesvirus, and a recombinantinfectious laryngotracheitis virus.
 9. The recombinant virus of claim 6,which further expresses a nucleotide sequence encoding at least oneantigen of an avian pathogen, or a nucleotide sequence biologicallyfunctionally equivalent thereto.
 10. The recombinant virus of claim 9,wherein said nucleotide sequence encoding at least one antigen of anavian pathogen or a nucleotide sequence biologically functionallyequivalent thereto encodes glycoprotein B of a Marek's disease virus.11. The recombinant virus of claim 9, wherein said nucleotide sequenceencoding at least one antigen of an avian pathogen or a nucleotidesequence biologically functionally equivalent thereto encodesglycoprotein B and glycoprotein E of a Marek's disease virus.
 12. Therecombinant virus of claim 9, wherein said nucleotide sequence encodingat least one antigen of an avian pathogen or a nucleotide sequencebiologically functionally equivalent thereto encodes glycoprotein B,glycoprotein E and glycoprotein I of a Marek's disease virus.
 13. Therecombinant virus of any claim 9, wherein said recombinant virus is arecombinant avipoxvirus or a recombinant avian herpesvirus.
 14. Therecombinant avian herpesvirus of claim 13, wherein said recombinantavian herpesvirus is serotype 1, 2, or 3 of Marek's disease virus.
 15. Arecombinant virus that expresses a DNA sequence encoding a membraneglycoprotein of Marek's Disease virus, wherein said recombinant virus isselected from the group consisting of a recombinant poxvirus, arecombinant avian herpesvirus, a recombinant adenovirus, and arecombinant influenza virus.
 16. The recombinant poxvirus or avianherpesvirus of claim 15, wherein said DNA sequence is shown in SEQ IDNO:1, or is a biologically functionally equivalent of said DNA sequence.